Deposit Feeder Ptychodera Bahamensis (Hemichordata: Enteropneusta)

MARINE ECOLOGY PROGRESS SERIES Vol. 45: 127-136, 1988 - Published June 20 Mar. Ecol. Prog. Ser. l

Microbial food resources of the macrofaunal- deposit feeder Ptychodera bahamensis (Hemichordata: Enteropneusta)

Fred C. ~obbs''*,James B. ~uckert~

' Department of Oceanography.Florida State University, Tallahassee, Florida 32306, USA Department of Microbiology, Montana State University, Bozeman, Montana 59717, USA

ABSTRACT Biochemcal and traditional analyses were used to characterize the microbial food resources and digestive efficiency of Ptychodera baharnenas an enteropneust hemlchordate Sediment was collected from freshly extruded fecal casts and adjacent feedlng depressions There were no sigmficant differences between casts and depressions in medlan gram size percent silt-clay, denslty of total meiofauna and of nematodes and concentrations of chlorophyll a and phaeophytin Nematodes in casts had a median diameter greater than those in depress~ons Measures of total vlable microbial biomass were 30 '10 (total phospholipid, ester-linked fatty acids) and 49 % (extractible phospholipid phosphate) lower m casts Concentrahons of 33 fatty acids were lower in casts, indicahng that the hemchordate dlgests a wde variety of microorganisms Only 18 lw7c (as-vaccenic acid) was not lower in casts than in depressions Thls fatty ac~d1s characteristic of eubactena havlng the anaerobic- desaturase pathway, many of which are Gram-negative organisms P baharnensis either cannot digest this funchonal group of bactena or contnbutes gut m~crobescontaining 18 lo7c to sedlrnent passing through its alimentary canal

INTRODUCTION Cuckert et al. 1985). An organism's fatty acid profile is a phenotypic description of its biochemical capabilities Benthc ecologists have long been interested in the and provides investigators with a powerful, dis- microbial food resources of animals that ingest sedi- criminating tool. ment, yet it usually is difficult to define exactly what We have studied the deposit feeding of Pytchodera those resources are. Many investigations have been bahamensis, an enteropneust hemichordate abundant limited to non-speciflc analyses, e. g. ATP and organic inter- and subtidally along the Gulf Coast of the Florida carbon, from whlch it is impossible to ascertain diges- Panhandle, USA. P. bahamensis inhabits deep burrows tion of specific microbial populations. Direct counts can and its modes of feeding and defecation are analogous distinguish between bacteria and diatoms, but are of to those of arenicolid polychaetes and leptosynaptid little benefit in assessing other microbial groups. holothurians, i. e. it is a 'funnel feeder'. Previous inves- As an alternative, quantitative extraction of cellular tigations of feeding by other enteropneusts include components having rapid turnover permits an assess- those of Cori (1902), Stiasny (1910), Barrington (1940), ment of the viable microbial community's biomass and and Knight-Jones (1953). However, none of these pre- composition without the problen~s associated with vious studies has been microbiological in character. In direct enumeration or cultural methods (White 1986). In fact, most of what is known concerning microbiological particular, phospholipid, ester-linked fatty acids have aspects of bulk feeding by deposit feeders, i. e. inges- proven to be reproducible, capable indicators of a wide tion of tens to hundreds of grams of sediment per day, suite of microbes encountered in the environment (e. g. stems from studies of surface-dwelling holothurians (e. g. Yingst 1976, Moriarty 1982, Hammond 1983, ' Present address: Marine Sciences Research Center, SUNY Baird & Thistle 1986). For only one group of subsur- Stony Brook, Stony Brook, New York 11794-5000, USA face-bulk ingestors, the arenicolid polychaetes, is any

O Inter-Research/Printed in F R. Germany 128 Mar. Ecol. Prog. Ser. 45: 123-136, 1988

information available on microbes' potential as nour- pling because of its highly elusive nature and, when ishment. Hylleberg (1975) observed that ciliates, flagel- collected, its proclivity to fragment. lates, small nematodes, and some bacteria were In the course of subsequent work on Ptychodera, we digested by Abarenicola pacifica. Boon et al. (1978) observed substantial variability in the appearance of showed that Arenicola marina contained algal- and casts. Color, composition, and granulometry may differ bacterial-derived fatty acids, implying that microbes within and among casts. Two end members in a spec- are a food resource. Rijken (1979) demonstrated that trum of fecal casts may be defined. 'Light' casts are addition of bacteria or diatoms to sediments increased beige, contain very few particles less than 63 bm in growth of juvenile A. marina in short-term (2 wk) diameter, and have essentially no debris from vascular experiments. It was not clear, however, whether the plant detritus. Light casts, therefore, have an appear- worms utilized living microorganisms or non-living ance very similar to ambient surface sediments. 'Dark' organic matter. casts are almost black, contain many particles less than The specific intent of the present study was to 63 pm in diameter (up to 10°/~ by weight; F.C.D. deduce the microbial food resources and the digestive unpubl.) and are composed of varying amounts of capabilities of Ptychodera bahamensis. While tradi- detritus from vascular plants. Light casts have total tional analyses were included in the research (e, g. sulfide concentrations at barely detectable levels (0.02 granulometry and meiofaunal counts), emphasis was pm01 g-l), while dark casts have higher concentrations placed on the use of phospholipid fatty acids to charac- (0.13 pm01 g-l) (Kevin Carman, FSU Dept of Oceano- terize the worm's resources at the level of functio~al graphy, pers. comm.). groups of microbes. Unfortunately, this variability had not been dis- cerned when samples for this study were collected. Most fecal casts, however, are neither completely light MATERIALS AND METHODS nor completely dark. A continuum of shades typically is encountered, with only a few percent classified as end Organism. Ptychodera bahamensis (hereafter members. From a probab~listic basis, then, it was Ptychodera) lives in a burrow and feeds on surface unlikely that this study's samples included end mem- sediments. Feeding activity causes surface sediment to bers. In fact, inspection of archived sediment indicated slump and form a rounded depression, up to 5 cm deep, that none of the sampled casts were light- or dark-end at one end of its burrow (Fig. 1). At the other end of its members. We conclude that although we sampled a burrow, it deposits its fecal casts, structures that may range of cast types and introduced an unanticipated rise 5 to 6 cm above the sediment surface. The fecal source of variation, the data were not skewed, but casts are 3 to 4 mm thick, loosely coiled, and weakly represented central tendencies. held together by a very thin mucus covering. The Field methods. The study site is an intertidal sand feeding depression and the fecal cast are the initial and flat in St. George Sound, approximately 50 m offshore final components of the deposit-feeding system of from the Florida State University Marine Laboratory, Ptychodera. The worm itself is not amenable to sam- Florida, USA (29" 55.00 N, 84" 30.36 W). On 28 Janu- ary 1986, 5 fecal casts of intertidal Ptychodera were FEEDING FECAL CAST selected for sampling. Criteria for selection included DEPRESSION an observed issuance of a cast. After sampling each cast, a corresponding set of samples was taken from the closest feeding depression. Sediment was col- lected using an ethanol-cleaned spatula. Samples for chlorophylI a, phaeopigments, and lipids were trans- ferred to containers that were plunged into liquid nit- rogen for several minutes and transported on dry ice to the laboratory. There, samples for lipids were lyophilized immediately; those for photopigments were stored at -70 "C. Samples for granulometry were taken to the laboratory and frozen. Samples for meiofauna were fixed in the field with 10 % buffered Fig. 1. Ptychodera bahamensis.Diagrammatic representation formalin (final concentration ca 5%) to which Rose of the burrow and deposit-feeding system (after Stiasny 1910). Bengal sta~nhad been added. It was not possible to associate a feeding depression with a particular fecal cast, so the distance between the two 1s indi- Laboratory methods. Granulometry: Particle-size cated as unknown. See Duncan (1987) for a review of burrow distributions were estimated by wet-sievlng the sam- types made by enteropneusts ples through nested sieves (1000, 500, 250, 125, and 63 Dobbs & Guckert: M~crobialfood resour .ces of an enteropneust hem~chordate 129

pm) using paper-filtered (Whatman # 2) seawater. The range quantified, i. e. C14-C20. A Nelson Analytical fraction < 63 pm was collected by vacuum filtration on 2600 programmable laboratory data system was used. previously dried and weighed filters (Gelman, 0.4 pm). Where CO-elutionof FAME peaks occurred, quantifica- All fractions were rinsed with distilled water to remove tion was supplen~entedwith the information provided salts, washed into tared trays (except for the fraction by the polar column. Fatty acids were normalized to < 63 lim), and dried to constant weight at 105 'C. Grain pm01 g-' (dry weight) of sediment and fatty acid profi- sizes were expressed in phi units, where the phi value les were expressed as percentages of the total molar is the negative logz of the particle diameter in mm. recovery. Meiofauna:After 48 h of fixation, the samples were The separated FAME were tentatively based on washed on a 63 ,pm sieve, than transferred to 80% coelution or identical relative retention time of the ethanol. Samples were later examined using a dissect- sample FAME with standards obtained from Supelco, ing microscope and meiofauna were picked out. Inc., Applied Science Laboratories, Inc., and Nu-Chek Densities were expressed as number of organisms Prep. Tentative identifications were also based on pre- cm-3. In sorting the nematodes, it was noted that speci- vlously identified laboratory standards, including simi- mens from casts seemed larger than those from depres- lar samples taken from the study site (Guckert et al. sions. To examine this possibility, nematodes' body 1985). Further verification required gas chromatogra- diameters were determined. Using videomicroscopy, phy/mass spectrometry (GUMS) using a VG MM-16 measurements were made at the level of the mass spectrometer having a direct capillary inlet. Sepa- pharyngeal bulb, when discernible. Otherwise, diame- rate analyses were done on J & W non-polar DB-5 and ter was measured at the worm's widest point. polar DB-225 capillary columns (30 m X 0.2 mm) with Photopigments and filamentous cyanobacteria. Pig- this system. The identification of FAME was verified by ments were extracted and quantified following the the occurrence of characteristic fragmentation patterns procedure of Hylleberg & Galluci (1975), a modification reported previously (Ryhage & Stenhagen 1958, Dinh- of the method of Lorenzen (1970). Filamentous Nguyen et al. 1961). The diagnostic mass-spectral frag- cyanobacteria were picked from sediments sampled for ment of saturated FAME is m/e = 74, a result of a meiofauna. Densities were expressed as number of gamma-hydrogen migration to a double bond followed filaments cm-3. by a beta cleavage, a mass-spectral process commonly Lipids. All glassware was immersed for at least 12 h known as the McLafferty rearrangement (McCloskey in 6 M HCl, rinsed with deionized water, dried, and 1970). The degree of unsaturation of FAME was also rinsed with chloroform-methanol (1:1, v/v). Lipids were confirmed by mass-spectral interpretation (McCloskey extracted using a protocol modified by White et al. 1970), while the position and geometry of the double (1979a) from that of Bligh & Dyer (1959). Following bonds was inferred by the retention times on the 4 recovery in chloroform, replicate 3 m1 samples were different GC-columns stationary phases used. withdrawn from each sample for determination of Fatty acids are designated as total number of carbon extractible phospholipid phosphate (White et al. atoms:number of double bonds with the position of the 1979b). The remainder of the chloroform-extracted double bond closest to the aliphatic (a) end of the lipid was fractlonated on a silicic-acid chromatography molecule indicated with the geometry 'c' for cisand 't' column (Gehron & White 1983) into 3 general lipid for trans. The prefixes 'i' and 'a' refer to iso- and classes: neutral lipids, glycolipids, and polar lipids. The anteiso-branching, respectively. Other branching is fatty acids esterified to the phospholipids were methy- indicated as position of the additional methyl group lated by mild alkaline transesterification of the polar from the carboxyl (A) end, e. g. 10Me16:O. Cyclopropyl lipid fraction (Guckert et al. 1985). The resulting fatty fatty acids are designated as 'cy'. All polyunsaturated acid methyl esters (FAME) were separated and quan- fatty acids are presumed to be methylene interrupted tified by capillary gas chromatography using a Varian (e. g. 18:303 = 18:3a3,6,9). 3700 gas chromatograph with flame-ionization detec- Statistical methods. Although samples were col- tion. The separation system detailed in Guckert et al. lected in a randomized-block design, results were ana- (1985) was used, except that the gas chromatograph lysed using a non-parametric procedure for a com- was outfitted with dual capillary columns (Hewlett- pletely randomized design, i. e. Wilcoxon's rank-sum Packard non-polar cross methyl silicone and Supelco test (Hollander & Wolfe 1973). Statistical discrimination polar SP-2340, both 50 m X 0.2 mm) set up in a parallel lost by disregarding blocks was more than compen- configuration from a common injector (Sonchik & sated for by the increased power of the rank-sum test. Walker 1985). FAME were quantified based on peak Hurlbert (1984) considered that such a 'hybrid areas determined on the non-polar column relative to approach' should not raise the probability of Type I the area for an internal injection standard (C19:O). An error. Unless otherwise noted, a critical value of equimolar response was assumed for all FAME in the p = 0.05 was used. 130 Mar. Ecol. Prog. Ser. 45: 127-136, 1988

RESULTS Table 1. Characteristics of fecal casts (n = 5) and feedlng depressions (n = 5) of Ptychodera bahamensis. Values repre- Granulometry sent means (standard deviation). 'PLFA': phospholipid fatty acid. ' Difference between casts and depressions was signifi- cant Cp < 0.05) At the study site, the ambient sediment, represented by samples from feeding depressions, is a moderately Characteristic Depressions Casts well-sorted medium-fine sand, with a silt-clay content < 0.7 O/O by weight (Table 1). The corresponding sam- Median phi 2.24 (0.05) 2.19 (0.06) ples from fecal casts were not different in either O/O silt-clay 0.66 (0.34) 0 52 (0.40) respect. Thus, differences that occur in other parame- Meiofauna 73.8 (42.9) 59.0 (37.0) ters were assumed to be independent of granulometry. (organisms Nematodes 32.3 (9.7) 43.8 (22.7) (organisms cm-3) Chlorophyll a (pg g-') 2.59 (0.70) 2.58 (0.47) Meiofauna Phaeopigments (pg g-') 1.19 (0.41) 1.35 (0.53) Cyanobacteria 43.4 (36.8) 8.4 (15.8)' A total of 856 organisms was recovered. The abun- (filaments cm-3) Phospholipid phosphate 27.0 (17.3) 15.4 (3.6)' dance of total meiofauna was not significantly different (nmol g-l) in casts and depressions (Table 1). Similarly, Total PLFA (nmol g-l) 14.86 (2.16) 10.37 (3.76)' nematodes, which comprised 57% of the total meiofauna, were found in approximately equal densities in casts and depressions (Table 1). There was no difference in the ratio of nematodes to total meiofauna in casts and depressions. No differences in Feeding Depress~on density were detected in other taxa, which included Median = 25.93 n.196 platyhelminths, harpacticoid copepods, tardigrades, and annelids (data not shown). Measurements of nematodes' body diameters con- firmed what had been sensed while sorting; the median diameter of organisms collected from casts was greater than in those from depressions (p< 0.001) (Fig. 2).

Fecal Cast Median = 33.34 Photopigments and filamentous cyanobacteria n=293

Values of chlorophyll a were almost identical in casts "In t U+ and depressions (Table l), about 2.6 pg g-' sediment I I I I1 1991 I 20 40 60 80 100 120 (dry weight). Likewise, concentrations of phaeopig- BODY DIAMETER (pm) ments were similar in casts and depressions, 1.35 and 1.19 kg g-l, respectively. There was no statistically Fig. 2. Size-frequency distributions of nematodes' body significant difference between casts and depressions in diameters in samples from feeding depressions (n = 5) and chlorophyll a or phaeophytin, or in their ratio to one fecal casts (n = 5) of Ptychodera bahamensis. Arrow indicates another. On the other hand, filamentous cyanobacteria, the median value of each distribution apparently of one morphotype, exhibited significantly lower numbers in casts than in depressions (Table 1). Thirty-four PLFA were identified and quantified. Of these, 16:lo?c, 16:0, and 18:lto7c together contributed Lipids approximately 50 O/O of the fatty acid biomass; 10 other fatty acids were among those each comprising 2 % or Two estimates of total, viable microbial biomass were more (Table 2). These 13 fatty acids comprised more determined. Extractible phospholipid phosphate was than 80 % of all fatty acid biomass. All fatty acids and 49 % lower, and total phospholipid, ester-linked fatty their concentrations are listed in the Appendix. Only acids (PLFA) were 30 % lower in casts than in depres- two of the dominant fatty acids showed a significant sions (Table 1). To partition differences in biomass into difference in their relative contributions to microbial components of the microbial community, individual communities in casts and depressions. The straight- PLFA were analysed. chain 15:O exhibited a slightly lower value in casts Dobbs & Guckert h4icrobial food resources of an enteropneust hernichordate 131

(4.27 '10) than In depressions (5.94 %). The monoenoic an average concentration in casts 24 to 56 % lower than

18:107c contributed 8.51 O/O in depressions and 12.34 O/O in depressions (Fig. 3). In fact, 18:lo7c was the only in casts. fatty acid in which the average concentration was Every identified PLFA, except 18:lo17c, was more greater in casts than in depressions, although the abundant in depressions than in casts (Appendix). difference was negligible. Among the dominant fatty acids, all save 18:lo7c had Phospholipid fatty acids may be combined (by sum- ming their concentratlons) to deflne functional groups of microorganisms. For example, 18-, 20-, and 22-car- Table 2 Dominant fatty acids (> 2 of total phospholipid fatty acids In either location) In the feedlng depressions (n = 5) bon polyunsaturated fatty acids are restricted to and fecal casts (n = 5) of Ptychodera bahamensis Values eukaryotes (Shaw 1966), with some possible exceptions represent the mean (standard deviation) percent contributed (e. g. cyanobacteria: Kenyon 1972, Potts et al. 1987; by the fatty acid to total fatty acld blornass 'Difference deep-sea eubacteria: DeLong & Yayanos 1986). There- between casts and depressions was significant @ 0 05) 5 fore, long-chain polyunsaturated fatty acids represent a measure of the eukaryotic contribution to the entire Fatty acid Depressions-- Casts microbial community. This and other biomarker assignments are detailed in Table 3. Note that 18:lo7c 3.24 (0.52) was used as a biomarker for bacteria having the 3.00 (0.36) 5.07 (0.61) anaerobic-desaturase pathway (Fulco 1983), one of 2 5.94 (0.77) major pathways of unsaturated fatty acid synthesis in 17.74 (1 69) bacteria. 21.04 (3.88) In a manner similar to analyses of individual fatty 1.95 (0.20) 4.40 (0.87) acids, the relative contnbutions of functional groups of 2.67 (0.19) microbes were compared (Table 4). The group of fatty 8.51 (0.95) acids characteristic of prokaryotes, odd chains, 2.82 (0.38) branched chains, and cyclopropyl derivatives, contri- 2.76 (0.67) buted about 30 O/O to total fatty acid biomass. Polyun- 4.02 (0.67) saturated fatty acids, indicative of eukaryotes, accounted for about 8.5 '10. Eukaryotic markers may be divided into the 06 and 03 series, sometimes referred AVERAGE PE RC EN T DIFFERENCE to as the 'animal' and 'plant' series, respectively (Erwin -60 -SO -20 0 '30 1973). Each subgroup of eukaryotic markers contri-

buted about 4 O/O to total phospholipid biomass. Fatty acid biomarkers of eukaryotic photoautotrophs also

Table 3 Assignment of phospholipid, ester-hnked fatty aclds to functional groups of microbes

--Functional group- Fatty acid(s) Eukaryotes

w6 'animal' series o3 'plant' series 18:3w3,20:5~3~ Eukaryotic photoautotrophs 16:lw13t, 18.3~3,18.1wga,' Prokaryotes i15:0,a15:0,15:0,il?:0,a17:0, 17:0, 18:lw7c, cy19:O(o7,8), 10Me16:0, cy 17:O (o7,8)* Desulfobacter Bactena, anaerobic Fig. 3. Average percent difference in concentration of dorm- desaturase pathway nant fatty acids in feeding depressions and casts of Ptychodera bahamensls. Each bar represents the difference between the Sources: a Shaw (1966); Erwin (1973); c Jarnes (1968); means of depression and cast values. Negatlve values indicate * Harwood & Russell (1984); Dowling et al. (1986); ' Fulco a lower concentration in casts, positlve values a higher con- (1983) centration in casts 132 Mar. Ecol. Prog. Ser. 45: 127-136, 1988

Table 4. Relative contnbutions of functional groups of mi- centrations. For example, a difference between casts crobes in the feeding depressions (n = 5) and fecal casts (n = and depressions in the ratio of eukaryotic to prokaryo- 5) of Ptychodera baharnensis. Values represent the mean tic markers would suggest a disproportionate change in (standard deviation) percent contributed by the group to total fatty acid biomass. 'Difference between casts and depressions one of the groups as sediment is moved through the gut was significant @ 5 0.05) of Ptychodera. However, no such difference was observed (Table 5). In fact, the same result emerged for all combinations tested. No significant difference 1Group Depressions Casts I existed in the ratio of w6 to to3 markers. Ratios of trans Eukaryotes to cis fatty acid isomers, and of cyclopropyl-fatty acids w6 series to their corresponding cis isomers, also showed no w3 series difference. Each of these latter 2 ratios has been shown Photoautotrophs to increase in prokaryotes stressed by nutrient depriva- Prokaryotes Prokaryotes, less tion or anoxia (Guckert et al. 1985, 1986). Desulfobacter 18:107c DISCUSSION

There are at least 3 levels at which these results may

accounted for about 4 Oio. Fatty acids characterisiic of be interpreted. The simplest approach is subtractive, the sulfate-reducing bacteria Desulfobacter spp., 10Me i. e. to consider that differences between feeding 16:O and cy17:0, accounted for less than 3 %. In all the depressions and fecal casts represent the digestive above groups, the difference between depressions and activities of Ptychodera. We emphasize that casts were casts was not significant (Table 4). Only for 18:lo?c, sampled immediately after their appearance, so the biomarker for bacteria having the anaerobic- recruitment of microbes from adjacent sediment would desaturase pathway, was there a significant difference, not have affected their microbiological characteristics. a 45 O/O higher value in casts. A more complicated approach is to consider that while Concentrations of the functional groups' biomarkers Ptychodera may digest microorganisms, it may also (Fig. 4) indicated a pattern similar to that detected for contribute gut flora to the microbial community that individual fatty acids. Each functional group had a su~vesexposure to its alimentary canal. The most concentration in casts significantly lower than that in complex approach is to consider that the feeding feeding depressions, except for bacteria having the depressions represent a greater or lesser food resource anaerobic-desaturase pathway, whose biomarkers than Ptychodera actually ingests. For example, certain showed no difference. particles or vagile meiofauna may not be carried down Ratios of fatty acids (or of groups of fatty acids) may the feeding funnel into the worm's burrow, or, Ptycho- provide information unavailable from analyses of con- dera may feed on subsurface deposits having a resource spectrum different from that of surface sedi- ment. The above 3 approaches will be consulted hierar- AVERAGE PERCENT DIFFERENCE chically in discussing the results. -50 -30 -10 0 -10

Table 5. Ratios of indi\lidual fatty acid isomers and functional groups in the feedng depressions (R = 5) and fecal casts (n = 5) of Ptychodera baharnensis. Values represent the mean - (standard deviation) ratio. In none of the ratios was there a significant difference between depressions and casts

Ratio formed by: Depressions Casts

Eukaryotes/Prokaryotes 0.295 (0.064) 0.282 (0.028) anaer desat As above, less 18:lo)7c 0.416 (0.103) 0.492 (0.089) pathway u)6/w3 0.865 (0.098) 0.750 (0.239) 16:1~07t/16lo17c 0.033 (0.009) 0 036 (0.013) Fig. 4. Average percent dfference in concentrations of func- 18: lw7t/18.lo7c 0.056 (0.020) 0.044 (0.013) tional groups of fatty acids in feeding depressions and casts of cy17:O (o7,8)/16:10)7c 0.021 (0.005) 0.028 (0.019) Ptychodera baharnensis. As in Fig. 3. DSBACTER: Desul- cy19:O (w7,8)/18:lw7c 0.029 (0.008) 0.021 (0.009) fobacter spp. Dobbs & Guckert: Microbial food resources of an enteropneust hemichordate 133

Granulometry depth. He suggested that A, marina ingests subsurface meiofauna, but does not digest them. A complication Evidence from granulometric analyses indicated that with using Jensen's results as support for the second with respect to particle size, Ptychodera ingests sedi- hypothesis is that the unusual nematodes he found in ments non-selectively at the study site. In other species casts were thiobiotic (deeper-dwelling) organisms, of enteropneusts, non-selectivity has also been which he (Jensen 1986, 1987) has shown are more reported (Cori 1902, Knight-Jones 1953, Suchanek & slender than oxybiotic (surface-dwelling) species. Colin 1986), although the ciliary feeding mechanisms Resolution of these 2 hypotheses awaits (at least) common to the group provides them with the capability specific identification of the nematodes involved. of rejecting oversized particles prior to ingestion (Barrington 1965). Photopigments and filamentous cyanobacteria

Meiofauna Passage through the gut of Ptychodera did not alter the sediment's concentration of chlorophyll a, phaeo- Meiofaunal organisms and their largest subset, pigments, or their ratio to each other. Therefore, nematodes, exhibited no significant differences in although diatoms and other photosynthetic organisms density between feeding depressions and fresh fecal are abundant at the study site, the enteropneust casts. Similarly, the ratio of nematodes to total appeared not to utilize them based on results of pig- meiofauna was not significantly different. Thistle ment analyses (see discussion on PLFA for a contradic- (1980) found that harpacticoid copepods were 7 times tory conclusion and resolution of the apparent discre- less abundant in fresh fecal mounds of subtidal pancy). The lower number of cyanobacterial filaments Ptychodera than in adjacent surface sediment. In the in casts relative to depressions may indicate selective present study of intertidal Ptychodera, so few harpac- digestion of a subset of the photosynthetic community. ticoids were collected that a comparison was not pos- Alternatively, the limited motility characteristic of sible. Duncan (1984) demonstrated that nematodes filamentous cyanobacteria, coupled with a positive escaped from collapsing feeding depressions of phototropism, may be sufficient to keep most of them Balanoglossus aurantiacus, and speculated that differ- from being buried in the worm's feeding depression. ences in mobility among taxa would determine the Thus, they would tend not to be ingested and their effects of the hemichordate's bioturbation on the density in fecal casts would be relatively low. meiobenthic community. No results consistent with Duncan's observations were found in the present study. Given the apparent non-selective ingestion of sedi- Lipids ment particles and accompanying meiofauna by Ptychodera, it is intriguing that the median diameter of If results of this study are approached subtractively, nematodes in fecal casts was 22 O/O larger than in feed- the conclusion is that Ptychodera digests one-third to ing depressions (Fig. 2). Two hypotheses emerge. In one-half the biomass in ingested sediment. These val- the first, small nematodes are digested, but not to the ues are typical of utilization of microbes by deposit extent that the density of nematodes significantly feeders (review by Lopez & Levinton 1987). Further- decreases during passage through the alimentary more, the hemichordate apparently digests a wide vari- canal. Hylleberg (1975) found that Abarenicola ety of microorganisms with approximately equal facil- pacifica, a polychaete annelid that feeds in a manner ity. Findlay (1986) studied the same population of similar to Ptychodera, disproportionally digested small enteropneusts and reported concentrations of muramic nematodes. Nothing is known concerning the digestive acid, an indicator of prokaryotic biomass, to be approxi- efficacy of Ptychodera, but it probably has a suite of mately 60 % lower in freshly extruded casts than in digestive enzymes. Barrington (1940) reported amy- ambient sediments. Findlay's measurements of phos- lase, lipase, and protease activity in the gut of pholipid phosphate and total PLFA also were lower in Glossobalanus minutus, another enteropneust. casts, but not significantly so. In the second hypothesis, large nematodes are In the present study, only 18:1w?c apparently was ingested disproportionally to their occurrence in feed- unaffected by the digestive capabilities of the ing depressions. Either Ptychodera selectively ingests hemichordate. The fatty acid is characteristic of, but not large nematodes or they are more abundant in sedi- unique to, eubacteria having the anaerobic-desaturase ment beneath feeding depressions. In fecal casts of pathway, many of which are Gram-negative organ- Arenicola marina, Jensen (1987) found 3 species of isms. It is intriguing that one functional group of bac- nematodes not found in sediments less than 8 cm in teria apparently is unaffected by Ptychodera's alimen- 134 Mar. Ecol. Prog. Ser. 45: 127-136, 1988

tary canal while other bacteria and eukaryotes are ment constitutes the main food resource of Ptychodera. digested. An appealing explanation Lies in the context Thus, at least for the present study's samples, this third of biofilms (Costerton et al. 1987). If bacteria having level of interpretation seems not to apply. the anaerobic-desaturase pathway characteristically In 2 cases, results from biochemical aspects of ths surround themselves with a more extensive, or more study apparently were not supported by those from resistant, exogenous mucopolysaccharide, then they traditional analyses. First, counts of meiofaunal organ- would be protected from the digestive activity of isms &d not differ in casts and depressions, yet fatty Ptychodera. acid biomarkers of eukaryotic animals exhibited sig- A less parsimonious level of interpreting the fatty nificantly lower concentrations in casts. Note that acid data is to consider that enrichment of 18:1w?c in meiofaunal organisms retained on a 63 km sieve consti- casts results from colonization of ingested sediment by tute only a subset of all eukaryotic, heterotrophic gut microbes (or at least, by microbes occurring in the organisms whose membrane lipids were extracted. gut). That is, ambient, surface-dwelling microbes con- Thus, the apparent discrepancy between conventional taining 18:lw?c may be digested to a degree compar- and biochemical approaches illustrates the more com- able to other microorganisms, but gut microbes con- prehensive nature of the lipid analysis. Note also, how- taining 18:lo7c are added to sedlment within the ever, that phospholipid fatty acid biomarkers specific alimentary canal. Since the concentration of 18:lw7c for taxa of meiofauna are not yet known, if indeed they did not vary significantly between casts and depres- exist. Thus, hypotheses concerned with specific groups sions, this more-complex hypothesis requires mainie- of meiofauna are, at present, not testable using Lipid nance of a balance between digestion of ambient analyses. sedimentary bacteria and adltion of gut bacteria. Second, while concentrations of chlorophyll a and A final level of interpretation is to consider that the phaeopigments did not differ between casts and depre- data result from Ptychodera's ingestion of subsurface ssions, fatty acid biomarkers of eukaryotic, photo- sediment in addition to sediment from the feeding synthetic organisms had significantly lower concen- depression. If so, the worm utilizes a food resource in trations in casts. In this study, therefore, pigment con- addition to that defined and measured in this study. centrations may not have reliably indicated digestion of However, since microeukaryotes and meiofauna are photosynthetic microorganisms. concentrated in the upper several cm of sediments (Fenchel & Straarup 1971, Fleeger & Decho 1987), and Acknowledgements. We thank D. Balkwill, W Burnett, P. because the relative proportions of eukaryotic fatty LaRock, E. Powell. D. Thistle, D. White, and anonymous acid biomarkers did not differ between casts and reviewers for their comments on an earlier version of this depressions (Table 4), we consider that surface sedi- manuscript. We appreciate the openness of D. Thistle, D.

Appendix. Fatty acid profiles in the fecal cast of Ptychodera bahamensis and in the closest feeding depression. Values are in pm01 fatty acid P g-' sediment (dry wt) and P represent the mean (4 1 standard deviation) of 5 samples Fatty acid Feeding depression Fecal cast Fatty acid Feeding depression Fecal cast

i14:O 65 + 10 25 -C 5 17:lw8c 643 f 94 425 + 178 14:O 477 + 76 296 + 194 17:lw6c 143 f 19 98 f 36 i15:O 443 + 72 194 2 24 cyl7:O 55 f 15 4055 a15:O 748 +110 413 +39 17:O 293 ? 26 179 k 48 15: 1 94 2 12 55 +35 18:3~06 66f 12 40 f 15 15:O 870 4 43 466 +231 18:2w6 122 5 28 72 * 33 i16:O 23 5 4 14 -C 2 18:3w3 91 f 34 51 k 13 16:lwgc 167 2 25 114 e 38 18:lwgc 399 f 78 266 f 48 16:lwgt 69 f 7 41 +13 18:lw7c 1257 +177 1300 f 606 16:lo7c 2638 f 495 2004 +1073 18:lw7t 68 f 13 58 f 28 16:lo7t 85f 11 61 f 19 18:O 415 f 56 234 f 39 16:lw5c 103 4 33 47 ? 6 br19:l 51 f 10 27 4 8 16.lw13t 134 4 35 101 2 46 cy19.0 37 f 14 23f 5 16:O 3168 4 879 2313 +970 20:4w6 419 + 146 233 f 32 10Me16:O 287 +. 34 193 f 34 20:5w3 606 +169 453 +171 i17:O 222 f 59 108 f 21 20:O 79 f 30 52 f 7 f a17:O 148 2 67 72 61 Unidentified 286 54 249 f 158 f + i17.1~7 81 10 49 f 21 fatty acids Dobbs & Guckert: Microbial food resou rces of an enteropneust hemichordate 135

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This article was presented by Dr S. Y Newell; it was accepted for printing on March 18, 1988