Phorbin steryl ester formation by macrozooplankton in the Sargasso Sea

Org. Geochem. Vol. 24, No. 5, pp. 581-585, 1996

Pergamon PII: S0146-6380(96)00073-3

Copyright© 1996 ElsevierScienceLtd Printed in Great Britain.All rights reserved 0146-6380/96 $15.00 + 0.00

NOTE Phorbin steryl ester formation by macrozooplankton in the Sargasso Sea L I N D A L. K I N G and STUART G. W A K E H A M Skidaway Institute of Oceanography, 10 Ocean Science Circle, Savannah, GA 31411, U.S.A. (Receh~ed 29 March 1996; returned to author for revision 17 May 1996; accepted 18 June 1996)

Abstract--Plankton sampling during a recent cruise in the Sargasso Sea provided numerous gelatinous macrozooplankton (salps) whose large gut contents afforded an opportunity to investigate the formation of phorbin steryl esters (PSEs) during zooplankton grazing. Sufficient material was obtained from about 50 salps to allow detailed determination of distributions and carbon stable isotope compositions of free sterols and PSE-sterols. Phorbin steryl esters were abundant in the gut contents of the salps but were absent from a large volume (8000 1) sample of suspended particulate matter (SPM), the presumed dietary material of the salps. Free sterols in salp gut contents and in SPM had similar isotopic ratios suggesting that indeed the sterols in SPM might be a major source of sterols in the gut contents, but PSE-sterols were enriched in ~C by 1-2%o compared to free sterols, indicating a trophic fractionation during PSE formation. These results further strengthen the role of zooplankton grazing on phytoplankton in PSE production. Copyright © 1996 Elsevier Science Ltd Key words--phorbin steryl esters, salps, Sargasso Sea, carbon stable isotopes, sterols, zooplankton.

suspended particulate matter, grazing, chlorophyll degradation products, pyropheophorbide-a

INTRODUCTION Phorbin steryl esters (PSE's) have been identified in a variety of aquatic environments, (Eckhardt et al., 1991; King and Repeta, 1991, 1994; Prowse and Maxwell, 1991; Eckhardt et al., 1992; Pearce et al., 1993). Based on distributions of phytoplanktonderived sterols in PSEs and the specificity of the acid moiety (pyropheophorbide-a) as a degradation product of algal chlorophyll-a, King and Repeta (1991, 1994) proposed that esterification occurs during zooplankton herbivory, i.e. their grazing on phytoplankton. In contrast, Eckhardt et al. (1991, 1992) favored esterification during senescence following phytoplankton blooms, citing the presence of PSEs in a plankton sample from the Baltic Sea but their absence in algal cultures. Several laboratory studies evaluate compositional changes of algal pigments during algal senescence and zooplankton grazing but did not mention PSEs (Daley, 1973; Shuman and Lorenzen, 1975; Ziegler et al., 1988; Head and Harris, 1992; Spooner et al., 1994). This oversight may have been due partly to limited availability of analytical tools. For example, Downs (1989) reported that zooplankton fecal pellets contained unidentified pigments which are now seen to have chromatographic properties corresponding with PSEs. Recent laboratory feeding experiments by Harradine et al. (1996) specifically addressing algal pigment alteration and PSE formation support the 581

zooplankton grazing pathway. Regardless of the mechanism by which PSEs are produced, incorporation of phytoplankton sterols into PSEs and the apparent stability of PSEs have been used to argue that the distribution of sterols in sedimentary PSEs may reflect the phytoplankton community structure in the surface ocean at the time of PSE production, possibly better than free sterols (King and Repeta, 1991, 1994; King, 1993). Most of the organic matter produced by phytoplankton passes through herbivorous zooplankton and microbes; some fraction of the material ingested by herbivores is transported to the sediment as fecal pellets (Wakeham and Lee, 1993). Although the presence of PSEs in sediment trap samples containing fecal pellets (King and Repeta, 1994) implicates zooplankton grazing in PSE formation, it is difficult to collect sufficient fecal material in the field to analyze for PSEs. During a recent cruise in the Sargasso Sea, we encountered numerous gelatinous macrozooplankton (salps) 2-5 cm in length and with full guts which might provide enough "protofecal" material for molecular and isotopic analysis of PSEs. Distributions and isotopic compositions of free and PSE-sterols in the salp gut contents were compared with suspended particulate matter, the likely food source for the salps. Our goal was to evaluate the source of PSEs, and further substantiate the possibility that these compounds record phytoplankton distributions.

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Note METHODS

Salp-gut contents were collected by excising guts from salps (approx. 50 large solitary asexually reproducing members) collected in net tows down to 100 m over a 5-night period (August 30-September 3, 1993). Suspended particulate matter (SPM) was collected in the Sargasso Sea by filtering 8000 I of -20-m deep seawater through 293 mm glass fiber filters (Gelman type A/E) over three consecutive days (September 1-3, 1993). Gut materials and SPM samples were frozen until analyzed. The SPM sample was Soxhlet-extracted using acetone followed by methylene chloride; the tissue sample was sonically extracted with acetone followed by methylene chloride. Following addition of 5% NaCI, the extracts were extracted with 30% (v/v) hexane/diethyl ether and the organic layers were combined and dried over Na2SO4. Free sterols were isolated from 5% of each extract by SiO2 column chromatography. Phorbin sterol esters were concentrated from the remaining extract on Merck Kieselgel 60 TLC plates developed with 25% acetone/hexane (v/v). Phorbin steryl esters were scraped from the plate, eluted with acetone, and isolated from the concentrate by HPLC (tR = 30-60 min) on an Adsorbosphere C~s column by elution with 20% n-propanol/methanol (v/v). The PSE's were further purified by SIO2, hydrolyzed in 5% H2SO4/methanol (wt%), partitioned into ethyl acetate/hexane and dried over Na2SO4. Prior to gas chromatographic analysis, sterols were derivatized to trimethylsilyl ethers with BSTFA/ acetonitrile. Gas chromatography was performed on a Carlo Erba 4160 gas chromatograph with a 25 m x0.25 #m i.d. DB-5 column and an on-column injector using H2 as carrier gas. The column was programmed from 120'~C to 310"C at Y C/min. Mass spectra were collected on a Hewlett Packard 5890 GC/Finnigan lncos 50 MS using a DB-5 column, on-column injection, and helium as carrier gas. Carbon isotope ratio determinations (relative to the PDB standard) were made on a Finnigan 252 isotope ratio MS coupled to a Varian GC with a 50 m HP Ultra-1 column. The GC was pr~ogrammed from 60°C to 200-~C at 20~C/min a n d 200r'C to 320C at 2'~C/min. The combustion reactor contained a Cu/PtO2 catalyst heated to 84OC. Data were corrected for isotopic shifts due to the derivatizing agent based on analyses of androstanol before and after addition of the trimethyl silyl group.

RESULTS AND DISCUSSION

Salps are a variety of planktonic tunicates, gelatinous macrozooplankton. They are non-selective filter feeders (Madin, 1974; Silver, 1975) that filter particles continuously (Wiebe et al., 1979) through a mucous net as they swim. The net retains 100% of particles larger than 4 ~tm and may retain particles as

small as 1 #m (Kremer and Madin, 1992). Salps produce fecal pellets within 16 h of feeding and their fecal pellets may play an important role in the vertical transfer of organic matter between surface waters and sediments (Madin, 1982; Wakeham and Canuel, 1988). As with many zooplankton, salps migrate vertically dielly, but most food consumption occurs during nocturnal hours in surface waters (Purcell and Madin, 1991). Though the depth of vertical migration may vary between 100 and 500 m for different species, salps are usually found within the upper 30-50 m during the night (Purcell and Madin, 1991). The feeding characteristics of salps lead us to presume that by analyzing suspended particulate material from near-surface seawater, we might reasonably sample the dietary source for the salps. Constraints on shipboard activities meant that seawater filtration occurred during the day and salp collection at night. The SPM sample contained a distribution of free sterols typical of mixed oceanic plankton, with cholest-5-en-33-ol G (letters refer to sterol identifications found in Fig. l) and 24-methylcholesta5,22E-dien-33-ol K as major constituents (Fig. 1). Although a few small zooplankton might have been present in such a large volume sample, the sterol distribution is strongly indicative of a dominance by phytoplankton. Excised salp guts contained a similar distribution of free sterols (Fig. 1), although the relative abundances of cholest-5-en-3fl-ol G and 24-methylcholesta-5,22E-dien-33-ol K were reversed compared to the SPM. Stanol/stenol ratios (A°/A s, A2"-/A ~22) were similar in both samples (0.1-0.3), also typical of phytoplankton (Nishimura and Koyama, 1977; Volkman et al., 1981). We were unable to detect PSE's in the suspended particle sample which we analyzed in total as one injection by HPLC. We conclude that an upper limit for a PSE concentration would be about 1 pg/l (10 ng/8000 l). In the salp-gut material, however, PSEs were present. PSE-sterols were measured in the gut material at a concentration of about 3% of that of the free sterols. The dominant sterols in the PSEs of the gut material were cholest-5-en-3fl-ol G, 24-methylcholesta-5,22E-dien-3fl-ol K, and 24-ethylcholest-5en-3/~-ol U (Fig. 1). Stanol/stenol ratios for PSE-sterols in gut material ranged from 0.1-0.3. The absence of PSE's in the SPM is consistent with previous findings. King (1993) did not find PSE's in SPM from the Black Sea, although sample sizes in that study were on the order of 20 1 as compared to 8000 1 here. If PSEs had been present in the Black Sea SPM in concentrations similar to their sedimentary concentrations relative to other pigments, they should have been found. Murray et al. (1986) provide HPLC chromatograms of SPM which show peaks in the region where PSEs were subsequently found to elute (King and Repeta, 1991). However, we cannot determine from available data whether these peaks are due to PSEs.

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Fig. 1. The distribution of (a) free sterols in suspended particulate matter, (b) free sterols in salp guts, (c) PSE-sterols in salp guts taken from the Sargasso Sea. Numbers above peaks are carbon stable isotope ratios in %0. A 24-norcholesta-5,22E-dien-3,8-ol, B 24-nor-5c~(H)-cholest-22E-en-3,8-ol, C 27-nor-24-methylcholesta-5,22E-dien-3,8-ol, D 27-nor-24-methyl-5cc(H)-cholest-22E-en-3,8-ol, E cholesta5,22E-dien-3,8-ol, F 5~(H)-cholest-22E-en-3,8-ol, G cholest-5-en-3,8-ol, H 5~(H)-cholestan-3,8-ol, ! cholesta-5,24(28)-dien-3,8-ol, K 24-methylcholesta-5,22E-dien-3,8-ol, L 24-methyl-5~(H)-cholest-22E-en3,8-ol, M 24-methyl-5~(H)-cholesta-5,24(28)-dien-3,8-ol, N 24-methylcholest-5-en-3,8-ol, O 24-methylcholesta-24(28)-dien-3,8-ol, P 24-methyl-5~(H)-cholestan-3,8-ol, Q 23,24-dimethycholesta-5,22E-dien3,8-ol, R 23,24-dimethycholest-22E-en-3,8-ol, S 24-ethylcholesta-5,22E-dien-3,8-ol, T 24-ethyl-5~(H)cholest-22E-en-3,8-ol, U 24-ethylcholest-5-en-3,8-ol, V 23,23-dimethylcholest-5-en-3,8-ol, W 24-ethyl5~(H)-cholestan-3,8-ol, DI 4~,23,24-trimethyl-5~(H)-cholest-22E-en-3,8-ol.

Free sterols in SPM and in salp-gut contents and PSE-sterols in gut material have marked similarities. Linear regressions of suspended particulate free sterols vs salp gut free sterols and of SPM free sterols vs salp gut PSE-sterols both yielded excellent correlation coefficients, r 2 = 0.94. Thus there appears to be a close coupling of free sterols in the SPM and free sterols in the gut contents (i.e. SPM is the food

source of the salps) and of free sterols in the SPM and in PSEs of the gut contents (i.e. sterols in SPM are incorporated into PSEs). There does not appear to be preferential enrichment of PSEs within the guts of the salps since we were unable to detect PSEs in SPM from a very large volume of seawater. Stable carbon isotopic compositions of free and PSE-sterols were determined to try to identify the

584

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source of the sterols in the PSEs or to at least link our SPM and salp-gut samples. 6~3C-Values for free AS-stenols in the suspended particles and in the salp guts ranged from - 2 6 to -29%o (Fig. 1). Several compounds yielded high standard deviations in their isotopic ratios which may indicate co-elution with other components. In general, however, both samples gave similar 6~3C-values for a given sterol (within one standard deviation). With the exception of two sterols, 24-methylcholesta-5,24(28)-dien-3fl-ol M and 24-methylcholest-5-en-3fl-ol N, each of the PSE stenols was 1-2%o enriched in 13C relative to their isotopic composition in either the suspended free or salp-gut free sterol pools. This is particularly well demonstrated for cholesta-5,22-dien-3fl-ol E, cholest5-en-3fl-ol G, 24-ethylcholest-5,22-dien-3fl-ol S, and 24-ethylcholest-5-en-3fl-ol U which have standard deviations of +1%o or less. Isotopic similarities between free sterols in SPM and salp-gut contents suggest a related planktonic source, whereas variations between salp-gut free and PSE sterols could reflect isotope fractionation during PSE formation or natural variability between samples. Our data set size prevents us from evaluating these options. In a few cases, we were able to obtain isotopic compositions of/l°-stanols in the salp gut free sterols (e.g. 5~(H)-cholestan-3fl-ol H and 24-methyl-5~t(H)cholest-22E-en-3fl-ol L). High standard deviations for isotopic compositions of stanols are most likely due to sample size limitations since base-line resolution was achieved between dS-stenol-d°-stanol pairs. The A°-stanols were significantly depleted in ~3C relative to their corresponding AS-stenols. This observation raises the question of whether there is a direct precursor-product relationship between A 5stenols and A°-stanols (Nishimura and Koyama, 1977) or whether the pairs of compounds have distinct biosynthetic sources. Two processes for formation of PSEs in the water column have been discussed in the literature. Eckhardt et al. (1991, 1992) suggested PSEs are formed during senescence following phytoplankton blooms, and Kowalewska (1994) recently concurred. The senescence pathway is based partly on the finding of PSEs in a plankton sample collected in the Baltic Sea (Eckhardt et al., 1992), but the use of a 60 pm plankton net will undoubtedly collect numerous zooplankton whose guts may contain PSEs. While we cannot rule out the senescence pathway completely, if it were important, we might have expected to find PSEs in the Sargasso Sea SPM sample we analyzed. We do not believe that the salp-gut sample we analyzed represents merely a selective concentration (of at least I-2 orders of magnitude) of PSEs from SPM. For example, salps filter 10-100 ml of seawater per min (Harbison and Gilmer, 1976; Harbison and McAlister, 1980) and defecate 4-8 fecal pellets per day (egestion rates of 400-800 pgC/d in 100 pg pellets; Madin, 1982). If, for the approximately 50 salps sampled we assume a gut clearance rate of 4-8

per day, then our sample of gut contents represents 3-6 h of feeding per salp and an effective concentration of material from a total of 90-1800 1 of seawater for our entire salp sample which is many times smaller than the SPM sample. If the salps were ingesting the PSEs from their food source, we would have expected PSEs in the SPM sample. Evidence is mounting in favor of PSE formation in guts of herbivorous zooplankton. Previous field work demonstrated the presence of PSEs in sediment traps which contained zooplankton fecal pellets (King and Repeta, 1994). The recent laboratory investigation by Harradine et al. (1996) in which the diatom, Thalassiosira weissflogii, was fed to the copepod, Calanus helgolandicus, with PSEs being found in the copoepod's fecal pellets, lends additional support in that phorbin steryl esters were detected in copepod fecal pellets. Our field results showing PSEs in guts of salps, the absence of PSEs from suspended particulate matter which the salps ingest, and compositional and isotopic similarities between free and PSE sterols further strengthens the importance of PSE formation by herbivorous zooplankton in the natural environment. The biochemical pathway of PSE formation is still uncertain and a number of questions remain unresolved. Are PSEs formed by the action of digestive enzymes in the zooplankton's gut or by the action by gut microflora? Is chlorophyll-a converted to pyropheophorbide-a by demetallation and loss of the carbomethoxy group and phytol side-chain, a process known to occur during grazing (Downs, 1989; Roy et al., 1989), followed by esterification of the phorbin macrocycle and a phytosterol? Or is the phytol side-chain ofchlorophyll-a transesterified with a phytosterol followed by conversion pigment to its pyropheophorbide form through demetallation and loss of the carbomethoxy group from the pigment? If the latter were the case, we would expect to find pheophorbide-a steryl esters, which at this time have not been identified. We have shown that the distribution of sterols esterified to pyropheophorbide-a in salp-gut contents mimics well the distribution of sterols found in the food source, i.e., phytoplankton in the SPM. Likewise, Harradine et al. (1996) found similar sterol compositions between the diatom feedstock and PSE sterols in copepod fecal pellets. This type of compositional relationship is required if PSEs are to be useful as proxies for surface water phytoplankton community structure and paleoceanographic reconstructions (King and Repeta, 1994). On the other hand, the PSE sterol composition of the fieldcollected salp-gut sample is considerably more complex than that of the laboratory-generated fecal pellets, reflecting a more complex phytoplankton distribution in nature compared to the single-species feeding experiment. It thus remains to be determined how faithfully compositions of PSEs in partculate matter and sediments track variations in phytosterol

Note distributions in response to shifts in p h y t o p l a n k t o n c o m m u n i t y structures over time a n d space in n a t u r a l environments. Results o f King a n d Repeta (1984) for time-series sediment traps show temporal variations in the c o m p o s i t i o n of sterols in PSEs which were interpreted as resulting from changes in p h y t o p l a n k t o n species composition, but there has yet to be a synoptic study o f PSE c o m p o s i t i o n produced by h e r b i v o r o u s z o o p l a n k t o n a n d the sterol composition o f the p h y t o p l a n k t o n the z o o p l a n k t o n are grazing. Associate Editor--J. A. C U R I A L E Acknowledgements--We thank the captain and crew of R/V Weatherbird for help in collecting the Bermuda samples, and Dr M. Silver and Dr J. Nelson for assistance. Dr K. Freeman assisted in performing the isotopic analyses at the Pennsylvania State University Isotope Facility. This work was funded in part by National Science Foundation grants OCE-9101667 and OCE-9123420 (to SGW) and OCE9202082 to the Pennsylvania State University Isotope Facility. Fifty percent of this research was funded by the U.S. Department of Energy's (DOE) National Institute for Global Environmental Change (NIGEC) through the N1GEC Southeast Regional Office at the University of Alabama, Tuscaloosa (DOE Cooperative Agreement No. DE-FC03-90ER61010); financial support does not constitute an endorsement by DOE of the views expressed in this article. REFERENCES

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