MARINE ECOLOGY PROGRESS SERIES Vol. 292: 51–60, 2005 Published May 12 Mar Ecol Prog Ser

Nutritional associations among fauna at hydrocarbon seep communities in the Gulf of Mexico

Stephen E. MacAvoy1, 2,* Charles R. Fisher3, Robert S. Carney4, Stephen A. Macko2

1Biology Department, American University, Washington, DC 20016, USA 2Department of Environmental Sciences, University of Virginia, Charlottesville, Virginia 22903, USA 3Biology Department, Pennsylvania State University, University Park, Pennsylvania 16802, USA 4Coastal Studies Institute, Louisiana State University, Baton Rouge, Louisiana 70803, USA

ABSTRACT: The Gulf of Mexico supports dense aggregations of megafauna associated with hydro- carbon seeps on the Louisiana Slope. The visually dominant megafauna at the seeps — mussels and tube worms — derive their nutrition from symbiotic relationships with sulfide or methane-oxidizing bacteria. The structure of the tube worm aggregations provide biogenic habitat for numerous species of heterotrophic animals. Carbon, nitrogen and sulfur stable isotope analyses of heterotrophic fauna collected with tube worm aggregations in the Green Canyon Lease area (GC 185) indicate that most of these species derive the bulk of their nutrition from chemoautolithotrophic sources. The isotope analyses also indicate that although 2 species may be deriving significant nutritional input from the bivalves, none of the species analyzed were feeding directly on the tube worms. Grazing gastropods and deposit-feeding sipunculids were used to estimate the isotopic value of the free-living chemoau- tolithotrophic bacteria associated with the tube worms (δ13C –32 to –20‰; δ15N 0 to 7‰; δ34S –14 to –1‰). The use of tissue δ34S analyses in conjunction with tissue δ13C and δ15N led to several insights into the trophic biology of the communities that would not have been evident from tissue stable C and N analyses alone.

KEY WORDS: Hydrocarbon seeps · Chemosynthesis · Sulfur isotopes

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INTRODUCTION biotic bacteria similar to those in the hydrothermal sys- tem (Kennicutt et al. 1985). It was soon discovered that During trawls in the Green Canyon Lease area off the tube worms (Seepiophila jonesi and Lamellibrachia the coast of Louisiana in 1985, previously unknown luymesi) and clams (Calyptogena ponderosa, Luci- species of tube worms and bivalves were discovered noma atlantis and Vesicomya cordata) harbored sym- near areas of seeping hydrocarbons and reduced sulfur biotic sulfide-oxidizing chemoautolithotrophic bacteria gases (Kennicutt et al. 1985, Brooks et al. 1987). The and the dominant mussel (Bathymodiolus childressi) Green Canyon tube worm and mussel species resem- was symbiotic with methanotrophic bacteria (Childress bled the invertebrate species that had been discovered et al. 1986, Brooks et al. 1987, Gardinar et al. 2001). near hydrothermal vents in the late 1970s (Ballard Subsequently, another mussel was discovered (Tamu 1977, Corliss & Ballard 1977). The hydrothermal vent fisheri) that harbors chemoautolithotrophic sulfur- invertebrates contained symbiotic chemoautotrophic oxidizing symbionts (Fisher 1993). Free-living bacteria bacteria that oxidized reduced sulfur species for also abound in the seep environment, often forming energy (reviewed by Childress & Fisher 1992). large orange, yellow and white mats (MacDonald et al. Because the worms and bivalves found at the hydro- 1989). It is generally believed that chemosynthetic pri- carbon seeps were related to the hydrothermal vent mary production forms the base of the hydrocarbon invertebrates and occurred at high densities around seep community food web, although contributions the seeps, it was postulated that they contained sym- from photosynthetically derived organic matter may

*Email: [email protected] © Inter-Research 2005 · www.int-res.com 52 Mar Ecol Prog Ser 292: 51–60, 2005

occur (Page et al. 1990, Kennicutt et al. 1992, Conway et al. 2002) and the nutritional relations among the et al. 1994, Carney 1994, Fisher 1996, Pile & Young seep fauna are not well understood. 1999, Levin & Michener 2002). In this study, tissue δ13C, δ15N and δ34S analyses were The methane and other reduced carbon compounds used to investigate trophic linkages among the inver- in the shallow sediments at this site largely derive from tebrate fauna closely associated with tube worm seepage from deeper source rocks (MacDonald et al. aggregations located at hydrocarbon seeps off the

1990, Sassen et al. 1999). The H2S is apparently largely coast of Louisiana (550 m depth). There were 4 primary produced by anaerobic sulfate-reducing bacteria in objectives of this study: (1) To determine whether het- the shallow sediments and is linked to methane oxida- erotrophic seep fauna collected with tube worms tion and possibly the oxidation of higher molecular derived the bulk of their nutrition from chemo- weight hydrocarbons (Formolo et al. 2004, Joye et al. autolithotrophic or methanotrophic primary produc- 2004, Sassen et al. 2004), but some sulfides may also be tion. (2) to determine whether the symbiontcontaining associated with the oil reservoir seepage (MacDonald invertebrates were an important food source for et al. 1989). The sediments may release some CH4, heterotrophs closely associated with the vestimentifer- 2– + 34 H2S, S2O3 and NH4 into the water column (Conway ans. (3) to determine if analyses of tissue δ S values et al. 1994), however, these compounds are only abun- provided additional insights into trophic relations dant at or below the sediment–water interface (Julian among seep fauna, beyond what could be learned from et al. 1999, Freytag et al. 2001). The carbon sources for analysis of δ13C and δ15N. (4) To constrain the stable C, the thiotrophic and methanotrophic bacteria are differ- N, and S values of free-living seep bacteria consumed ent. The carbon source for the thiotrophic bacteria is by heterotrophic primary consumers. dissolved inorganic carbon (DIC), while the methan- otrophic bacteria use CH4 as both a carbon and energy source (Smith & Hoare 1977, Colby et al. 1979, Cary et MATERIALS AND METHODS al. 1988). Stable isotope analyses have played an important The Green Canyon Lease Area occupies approxi- role in understanding hydrothermal vent and hydro- mately 22 000 km2 of seafloor lying between the 200 carbon seep ecosystems. In fact, the first real evi- and 2400 m isobaths on the topographically and geo- dence that the nutrition of vent animals was based on chemically complex continental slope off the coast of local primary production rather than photosynthetic Louisiana. For the management of oil and gas devel- production were stable C and N analyses (Rau & opment, the area is subdivided into 4.8 × 4.8 km lease Hedges 1979, Rau 1981). Similarly, stable isotopes blocks. This study was conducted on communities were important in determining the carbon and from one of these lease blocks (GC 185; Bush Hill, energy sources for symbiont-containing hydrocarbon located at 27° 46.96’ N, 91° 30.46’ W, 540 to 580 m seep fauna (Childress et al. 1986, Brooks et al. 1987, b.s.l.). GC 185 is an active hydrocarbon seep area Fisher et al. 1993). Stable isotopes have also been a supporting chemoautolithotrophic communities that valuable tool for understanding trophic relationships have been studied for various purposes for more than among heterotrophic organisms within hydrothermal a decade (Brooks et al. 1987, Kennicutt et al. 1992, vent communities (Van Dover & Fry 1989, 1994, Fisher 1996, Sassen et al. 1999). Tube worms are the Fisher et al. 1994). Nitrogen isotopes are good indica- visually dominant symbiont-containing group at the tors of food chain position (an approximately 3.4‰ site, although mussels symbiotic with thiotrophic or enrichment per trophic level; Minagawa & Wada methanotrophic bacteria (Tamu fisheri and Bathy- 1984), and carbon and sulfur isotopes have been modiolus childressi, respectively) also occur here. shown to closely reflect an animal’s food source (Fry Methane and oil are actively seeping from the sedi- & Sherr 1984, Peterson et al. 1985, Peterson & ments in several places on Bush Hill. Bacterial mats Howarth 1987, Hesslein et al. 1989, 1991, Kline et al. and carbonate outcrops are abundant, as are shallow 1990, 1993, MacAvoy et al. 1998). The invertebrate gas hydrates (Nix et al. 1995, Sassen & Macdonald and vertebrate taxa collected and observed among 1997, Sassen et al. 1999). the chemoautolithotrophic animals at hydrocarbon Isotope analysis. Two tube worm aggregations were seeps in the Gulf of Mexico are a fairly diverse group, collected intact with the DSV Johnson-Sea-Link II including crabs, shrimp, isopods, gastropods, poly- (Harbor Branch Oceanographic, Fort Pierce, FL) using chaete worms and fish (MacDonald et al. 1989, Car- a hydraulically activated device that encloses the tube ney 1994, Bergquist et al. 2003). To date, stable iso- worms and associated fauna, and removes the entire topic analyses have only been reported for a limited aggregation (Bushmaster Jr., Bergquist et al. 2000). number heterotrophic species from the Gulf of Mex- One aggregation was dominated by larger individuals ico seeps (Brooks et al. 1987, Fisher 1996, MacAvoy with no recent tube worm recruits present and is here- MacAvoy et al.: Nutrition associations in hydrocarbon seep communities 53

after referred to as the older aggregation (BH-7 in N ≥ 3. Microsoft Excel 5.0 (Microsoft) and Statview SE Bergquist et al. 2002). The second younger aggrega- + Graphics (Abacus Concepts) were used for individ- tion (BH-1 in Bergquist et al. 2002) was dominated by ual statistical tests. small individuals, tube worm recruitment was recent or ongoing at the time of collection, and the mean tube worm age in the aggregation was calculated to be 3 yr RESULTS (Bergquist et al. 2002). Tissue samples were dissected at sea from all animals and were frozen until shipment Chemoautolithotrophic symbiont-containing to the stable isotope laboratory. The tissue type sam- invertebrates pled was muscle tissue for most invertebrates, excep- tions being the tube worms and mussels (plume tissue In 1998, 2 separate tube worm aggregations were and mantle tissue were used, respectively). Samples collected at GC 185 (Bush Hill). Two tube worm spe- arriving at the laboratory were thawed, dried at 60°C cies, Lamellibrachia luymesi and Seepiophila jonesi, for 3 d and homogenized by a mortar-and-pestle grind- were present in both collections, and the younger ing tool. Approximately 5 to 6 mg of tissue was used for aggregation also included the mussels Tamu fisheri δ34S measurements (1 to 2 mg was used for tube worm and Bathymodiolus childressi. The B. childressi mussel plumes owing to the high sulfur content) and 0.6 to is symbiotic with methanotrophic bacteria; however, it 1.0 mg was used for δ13C and δ15N measurements. A retains a functional gut and is capable of filter feeding Carlo Erba elemental analyzer coupled to a Micromass (Page et al. 1990, Pile & Young 1999). The other 3 Optima isotope ratio mass spectrometer (Micromass) symbiont-containing species harbor sulfur-oxidizing was used to obtain δ13C, δ15N and δ34S values (Fry et al. bacteria. 1992, Giesemann et al. 1994). The δ13C and δ15N values Tube worms were significantly 13C-enriched relative were determined concurrently and δ34S was deter- to both mussel species (Table 1). Bathymodiolus mined during separate analysis runs. childressi and Seepiophila jonesi δ15N values were not The isotope compositions are reported relative to significantly different from each other, but were signif- standard material and follow the same procedure for icantly different from Lamellibrachia luymesi and all stable isotopic measurements as follows: Tamu fisheri. L. luymesi were 34S-depleted relative to B. childressi (Table 1). T. fisheri δ34S values fell be- δxE = [(xE/yE) /(xE/yE) – 1] × 1000 (1) sample standard tween those of L. luymesi and B. childressi, however, T. where E is the element analyzed (C, N or S), x is the fisheri were not significantly different from either molecular weight of the heavier isotope, and y the group (0.1 < p > 0.05). lighter isotope (x = 13, 15, 34 and y = 12, 14, 32 for C, N and S, respectively). The standard materials to which the samples are compared are PDB for carbon, air N2 Heterotrophs for nitrogen and CDT for sulfur. Reproducibility of all measurements was typically 0.3‰ for δ34S and 0.2‰ Likely consumers of primary production (chemo- for δ13C and δ15N. Approximately 217 analyses were synthetic or photosynthetic) are grazing gastropods conducted using 84 individuals from 24 different and deposit feeders. Most grazing gastropods, which species. were collected from the young tube worm aggrega- Statistical analysis. Mann-Whitney U-tests were tion, were depleted in 13C relative to that typically seen used for 2 group comparisons and Kruskal-Wallace in Gulf of Mexico (GOM) particulate organic matter tests were used for all multiple com- parisons (α = 0.05). The Dunn proce- Table 1. Isotope values for invertebrates with symbionts collected at GC 185. dure was used to compare specific dif- Comparisons were made within the collection, not between the collections. Those with different letters are significantly different from each other (p ≤ 0.05) ferences among groups if Kruskal-Wallis indicated a significant δ13 δ15 δ34 difference (Rosner 1990). The Dunn Invertebrate C ± SD (N) N ± SD (N) S ± SD (N) procedure reduces the risk of Type 1 Old tube worm bush error inherent in multiple comparison Lamellibrachia luymesi –20.1 ± 1.2 (6)a 2.2 ± 0.4 (6)a –27.2 ± 5.8 (4)a,b techniques by increasing the Z-score Seepiophila jonesi –22.0 ± 0.4 (5)b 3.2 ± 0.6 (5)b –32.8 ± 3.0 (3)a,b needed to reject the null hypothesis as Young tube worm bush the number of individual groups Lamellibrachia luymesi –21.5 ± 1.2 (6)a 0.7 ± 0.7 (6)a –25.1 ± 2.4 (5)a,b (treatments) increases. The statistical Seepiophila jonesi –22.2 ± 2.3 (4)a 3.8 ± 1.3 (4)b Tamu fisheri –37.3 ± 0.9 (3)b –0.4 ± 1.8 (3)a– –16.9 ± 1.9 (3)a,b comparisons were only made when Bathymodiolus childressi –42.9 ± 1.5 (6)b 6.1 ± 1.2 (6)b –5.3 ± 1.4 (6)b,, individual groups (treatments) had an 54 Mar Ecol Prog Ser 292: 51–60, 2005

Bathynertia naticoidea Phascoloma turnerae S. jonesi L. lyumesi Munidopsis sp. A. stactophila Provanna sculpta Cataegis meroglypta E. canetae S. tanneri B. childressi T. fisheri 11 10 10 a a 8 9 6

N 8 N 15 15 δ

7 δ 4 6 2 5 0 4 3 -2 -34 -32 -30 -28 -26 -24 -22 -20 -50 -45 -40 -35 -30 -25 -20 -15 13 δ13C δ C Bathynertia naticoidea Phascoloma turnerae L. lyumesi A. stactophila E. canetae Provanna sculpta S. tanneri B. childressi T. fisheri 2 10

0 b 5 b 0 -2 -5

-4 S S

34 -10 34 δ

δ -6 -15 -8 -20 -10 -25 -12 -30 -34 -32 -30 -28 -26 -24 -22 -20 -50 -45 -40 -35 -30 -25 -20 -15 δ13C δ13C Fig. 1. δ13C vs. (a) δ15N and (b) δ34S for grazing gastropods Fig. 3. δ13C vs. (a) δ15N and (b) δ34S for a subset of invertebrates associated with a young tube worm bush at GC 185 closely associated with a young tube worm bush at GC 185

S. jonesi L. luymesi Munidopsis sp. (POM, –21.4 ± 1.4‰; Fry 1983, Macko et al. 1984, A. stactophila Phymorhynchus sp. δ13 10 although the tissue C values for 2 individuals were 9 a in the range of values reported for POM (Fig. 1). The 15 8 δ N values of the grazers ranged from 2 to 10‰, which 7 tended to be somewhat lower than particulate organic

N 6 matter (7.5 ± 0.8‰) and zooplankton (8.9 ± 0.9) in the 15 δ 5 NW GOM (Fry 1983, Macko et al. 1984). Sulfur in 4 grazers as a group was highly 34S-depleted relative to 3 POM (marine POM δ34S = 18 to 20‰; overlying GOM 2 bottom waters δ34S = 20.3‰; Aharon & Fu 2003), 1 -32 -30 -28 -26 -24 -22 -20 -18 -16 however, there was a 10‰ range among the 6 indi- δ13C viduals analyzed (–10 to 0‰) (Fig. 1). The deposit- S. jonesi L. luymesi Munidopsis sp. feeding, primary-consumer sipunculid Phascolosoma A. stactophila Phymorhynchus sp. 20 turnerae lives in the sediment at the base of the tube worm aggregation. P. turnerae in both tube worm b 10 collections were depleted in 13C and 34S relative to 0 values expected for photosynthetic POM. P. turnerae isotope values were similar to those seen in grazing -10 S

34 gastropods (Fig. 1, Table 2). δ -20 Most other invertebrate species captured within the 34 -30 tube worm aggregations were also S-depleted rel- ative to GOM POM (Table 2, Figs. 2 & 3). Nine of the -40 13 -32 -30 -28 -26 -24 -22 -20 -18 -16 other 14 species were C-depleted. The only indi- 13 34 δ13C vidual not depleted in either C or S relative to the Fig. 2. δ13C vs. (a) δ15N and (b) δ34S for a subset of invertebrates other heterotrophs was a hagfish, Eptatretus sp. (Table closely associated with an old tube worm bush at GC 185 2). The 2 other vertebrates captured were a conger eel MacAvoy et al.: Nutrition associations in hydrocarbon seep communities 55

Table 2. Isotope values (means ± standard deviation [N]) for hydrocarbon seep and the fact that stomach contents of organisms captured in Bushmaster collections at GC 185 animals that eat bacteria are almost impossible to characterize, especially Organism δ13C ± SD (N) δ15N ± SD (N) δ34S ± SD (N) hours after the animals are collected. Additionally, captured consumers Old tube worm bush often have a chance to eat damaged Gastropods animals in the collection containers, so Phymorhynchus sp. –22.2 ± 0.8 (6) 4.1 ± 0.6 (6) 4.6 ± 6.6 (4) Limpet –30.2 ± 2.5 (2) 9.6 ± 0.6 (2) 4.9 (1) the most recent meal may not be Worm-like taxa indicative of their normal food source Nemertean –23.1 (1) 7.5 (1) –6.1 (1) (authors’ pers. obs.). Crustacea Stable isotopes cannot tell you what Munidopsis sp. –26.8 ± 2.7 (7) 7.5 ± 1.6 (7) –4.4 (1) an animal has been eating; however Alvinocaris stactophila –20.6 (1) 5.4 (1) –0.3 (1) they can constrain the possibilities, Ayiielae shrimp –33.2 (1) 2.6 (1) and sometimes tell you what an ani- Atelecylidae crab –24.8 (1) 9.7 (1) mal has not been eating, as well as Fish Eptatretus sp. –20.9 (1) 11.5 (1) 12.2 (1) provide general information on food Dysommina rugosa –30.4 (1) 7.4 (1) sources. Because of the remoteness of Ophicthus cruentifer –33.4 (1) 7.4 (1) –13.3 (1) the communities we are studying, sta- Other ble isotopes are an excellent way to Phascolosoma turnerae –28.0 ± 0.5 (6) 6.3 ± 1.7 (6) –7.6 ± 3.8 (4) start, particularly with the addition of Sclerasterias tanneri –32.9 ± 0.5 (2) 5.5 ± 0.1 (2) δ34S to the more commonly reported Younger tube worm bush δ13C and δ15N. This study provides Gastropods basic information about what fuels the Provanna sculpta –31.5 ± 1.2 (2) 4.0 ± 0.8 (2) –9.9 (1) hydrocarbon seep communities in the Cataegis meroglypta –28.5 (1) 4.1 (1) Eosipho canetae –29.2 ± 9.1 (2) 6.2 ± 1.6 (2) –4.5 ± 6.3 (4) Gulf of Mexico. However, our results Bathynerita naticoidea –26.7 ± 4.8 (5) 8.2 ± 2.2 (5) –1.5 ± 1.4 (4) must be interpreted within the context Worm-like taxa of what is known about the life history Red nemertean –22.2 (1) –5.5 (1) –9.6 (1) and feeding biology of the taxa, and Polynoid, Harmothoe sp. –23.6 ± 0.1 (2) 5.7 ± 1.9 (2) –8.9 ± 2.4 (2) tested by further research. Polychaete (Amphinomidae) –40.9 ± 0.9 (3) 8.1 ± 2.0 (3) –9.7 ± 2.5 (3) Maldanidae (Nicomache sp.) –28.5 (1) 7.5 (1) –2.9 (1) Crustacea Munidopsis sp. –28.7 (1) 7.7 (1) –7.5 (1) Chemoautolithotrophic symbiont- Alvinocaris stactophila –30.4 ± 6.2 (4) 8.2 ± 1.1 (4) 0.9 ± 5.3 (4) containing invertebrates Other Phascolosoma turnerae –29.3 (1) 8.3 (1) –3.2 ± 3.5 (2) The tube worms in the collections Sclerasterias tanneri –35.6 (1) 5.5 (1) 4.2 (1) were on the 13C-enriched side of the range of values that have been reported for these species on the (Ophicthus cruentifer) and a cut throat eel (Dysom- Louisiana Slope (Brooks et al. 1987, Kennicutt et al. mina rugosa). The 2 eels had tissue stable isotope 1992). The fact that the tube worm species are 13C- values similar to many of the invertebrate heterotrophs enriched relative to the thiotrophic-symbiont-contain- collected with the same tube worm aggregations. ing bivalve (Tamu fisheri) collected at the same site is However, the hagfish was the most 13C-, 15N- and consistent with patterns seen in hydrothermal vent 34S-enriched of any animal captured (Table 2). communities. As with the hydrothermal vent tube worms and mussels, this difference is likely due to (1) the fact that the symbionts in the 2 groups use different DISCUSSION forms of RibuloseBisPhosphate carboxylase/oxygenase (that fractionate to different extents), and (2) differ- Very little is known about the feeding biology of ences in morphology and physiology resulting in dif- most deep-sea animals, particularly the relatively ferential dissolved inorganic carbon (DIC) supply and newly discovered fauna of hydrocarbon seeps and exchange (Fisher et al. 1990, Robinson & Cavanaugh hydrothermal vents. Much can be learned from behav- 1995, Robinson et al. 1998, 2003). It is also possible that ioral observations and stomach content analyses, but the 2 groups are taking up DIC that is quite different in these approaches are of limited use in these environ- δ13C. In addition to the DIC in the seawater, inorganic ments because of the difficulty of obtaining the former CO2 can be produced from methane oxidation and 56 Mar Ecol Prog Ser 292: 51–60, 2005

degradation of organic material in the sediments or at a difference suggests that T. fisheri and L. luymesi may the sediment–water interface. As a result, δ13C values be acquiring sulfide from different pools. T. fisheri sul- for DIC in shallow sediments as low as –27.3‰ have fide acquisition would be limited to the sediment– been reported for this site (Sassen et al. 1999). T. fish- water interface or at most a few centimeters deep into eri is a relatively small species that lives partially the sediments, and the trend of more 34S depletion in buried with its siphon very close to the tissues of L. luymesi supports previous reports of sul- sediment–water interface, where DIC δ13C values fide uptake by root tissue (Freytag et al. 2001). Bathy- would reflect input from both interstitial and seawater modiolus childressi from the same collection as T. fish- sources. The 2 tube worm species could take up DIC eri were on average even more enriched in 34S, which either across their plumes (a well-vascularized gill-like is consistent with sulfur acquisition via a depleted sul- gas exchange organ) or across their posterior ends fate pool by these methanotrophic symbiont-contain- (roots), which can be deeply buried in the sediment ing animals (average δ34S = –16.9 and –5.3‰ for T. (Julian et al. 1999, Freytag et al. 2001). In the case of fisheri and B. childressi, respectively, p = 0.02; how- Seepiophila jonesi the plumes are located at the sedi- ever, this was not significant using the Dunn procedure ment–water interface while Lamellibrachia luymesi for individual comparisons within a Kruskal-Wallace grows with its plume well above the sediment, up to a test). It should be kept in mind, however, that B. chil- meter or more above the sediment in older aggrega- dressi (and likely T. fisheri) are capable of filter feed- tions (Bergquist et al. 2003). L. luymesi tissue carbon ing and so any of their tissue stable isotope values was significantly enriched in 13C compared to S. jonesi could be affected by input from other sources (Page et in the older tube worm aggregation (Table 1). This is al. 1990, Pile & Young 1999). consistent with the plume being the primary DIC uptake organ and a relatively 13C-depleted DIC pool at the sediment surface interface as a result of methan- Heterotrophs otrophic activity in the sediments. The difference in δ13C values between species in the younger tube worm When all 3 stable isotopes are considered together, it aggregation was not significant, but showed the same is clear that all invertebrates collected in close associa- trend. The other mussel collected with one of the tion with the tube worm aggregations for this study are aggregations, Bathymodiolus childressi, harbors most likely obtaining the bulk of their nutrition from methanotrophic symbionts and its tissue δ13C values chemosynthetic production, as are 2 of the 3 fishes reflect the δ13C of methane at this site (–44 to –46‰, (Tables 1 & 2). In the cases where the δ13C and δ15N Sassen et al. 1999). values alone could not differentiate between input of As for carbon, differences between the tissue stable photosynthetic and chemosynthetic material (Alvino- N isotope values in the 2 species of tube worms sug- caris stactophila and Phymorhynchus sp. in the gest that they are either utilizing different chemical younger bush and a Nemertean and a polynoid worm species of N, tapping different pools of N, or discrimi- in the older bush; Table 2), very depleted δ34S values nating differently after acquisition of their N source. (relative to ambient bottom water sulfate [20.3‰]; We cannot distinguish between these possibilities at Aharon & Fu 2003) clearly indicate the importance of this time, but note that the different growth forms and local primary production to the nutrition of these spe- resultant plume positions with respect to the sedi- cies, although they are greatly 34S-enriched relative to ments, described above for the 2 species, may con- the tube worm tissues. These results are somewhat tribute to this difference. similar to those of Micheli et al. (2002) who found that There is thought to be little fractionation between at hydrothermal vents on the East Pacific Rise, preda- sulfide and sulfate or organic sulfur as a result of sul- tors closely associated with the tube worms derive a fide oxidation by chemoautolithotrophic bacteria majority of their nutrition from chemosynthetic mater- (reviewed in Canfield 2001). Therefore, the tissue δ34S ial. However, unlike some of the invertebrate hetero- values of animals with chemoautolithotrophic sulfur- trophs examined in Micheli et al. (2002), which would oxidizing symbionts probably reflect their reduced sul- consume tube worm tissue under their experimental fur source. Although the δ34S values of the 2 tube worm conditions, the chemosynthetic production consumed species were not significantly different, the averages by invertebrate heterotrophs at these cold seeps is were separated by 5‰, suggesting that they may be unlikely to include tube worms. The δ34S values tapping different sulfide sources. Further analyses are of these organisms could indicate a mixing of 34S- needed to verify this trend. The mussel Tamu fisheri depleted chemosynthetic production and 34S-enriched appears to be enriched in 34S compared to Lamelli- photosynthetic production, however, the δ34S values brachia luymesi in the same aggregation, although the are much too 34S-enriched for tube worm tissue to be a trend is not statistically significant. The trend towards primary source of nutrition. This is consistent with the MacAvoy et al.: Nutrition associations in hydrocarbon seep communities 57

observations of Kicklighter et al. (2004) who found that species, one can constrain the range of stable isotope hydrocarbon seep tube worms (Seepiophila jonesi and values in the free-living bacteria present on the tube Lamellibrachia luymesi) were unpalatable to predators worm tubes and in the sediment beneath the aggrega- such as mummichogs and the lesser blue crab. They tion. This analysis is based on 1‰ enrichment associ- propose that these tube worms either have chemical ated with carbon, no enrichment associated with sulfur compounds in their tissues that deterred feeding and 3‰ enrichment associated with nitrogen per (extracts of trophosome were unpalatable to both trophic level (Minagawa & Wada 1984, Peterson & predators) or that the toughness of the tubes deterred Howarth 1987). The stable isotope values of the 3 graz- predation. Lack of predation on tubeworms is also con- ing gastropods captured within the tube worm aggre- sistent with the long lives and great ages achieved by gation (Bathynerita naticoidea, Provanna sculpta and tube worms at these seeps (Bergquist et al. 2000, Cataegis meroglypta) suggest an isotope range for the Cordes et al. 2003). The only species collected along material consumed off of tube worm tubes of –20 to with the tube worm aggregations that is not likely to –32‰ C, 0 to 7‰ N, and –14 to –1‰ S (Table 1, Fig. 1). obtain the bulk of its nutrition from chemoautotrophic The elevated δ15N of some B. naticoidae relative to P. production is the hagfish Eptatretus sp. (Table 2). This sculpta and C. meroglypta may reflect B. naticoidae’s is consistent with the results of previous studies that ability to consume small animals as well as bacteria as examined the role of seep nutrition in a hagfish and a polychaete setae, crustacean legs and sponge spicules variety of other mobile benthic consumers in the Gulf have been reported as gut contents for this species of Mexico (MacAvoy et al. 2002, 2003). (Zande 1994, Zande & Carney 2001). Very little is The combined use of all 3 isotopes is also critical known about the ecology of P. sculpta (Waren & in evaluation of the possible role of the symbiont- Bouchet 2001) other than the fact that this small gastro- containing animals in the nutrition of the heterotrophic pod (usually 5 mm in length or less) is endemic to the fauna. Based on δ13C and δ15N values alone, one might Louisiana hydrocarbon seeps (Carney 1994) and that it erroneously conclude that the predatory/scavenging probably grazes in a similar manner as C. meroglypta gastropod Phymorhynchus sp., and perhaps some indi- (Waren & Bouchet 1993). Interestingly the tissue δ13C viduals of the predatory Eosipho canetae and the graz- and δ15N values of P. sculpta individuals grouped very ing Bathynerita naticoidea, were feeding primarily on tightly around –32‰ and 4‰ respectively, and the the tube worm-derived carbon and nitrogen. However, 1 individual analyzed for δ34S, was very highly the very large difference between the tissue δ34S val- depleted in 34S. Either this species is feeding very ues of the tube worms and these consumers indicate selectively on a specific population of the free-living that this is very unlikely, although a mixed diet of tube bacteria present, or perhaps it has its own symbionts. worms and surface-derived material is consistent with The tissue δ13C, δ15N and δ34S values of the deposit- the isotope data (Fig. 2a). Taken together, the δ13C, feeding sipunculid Phascolosoma turnerae suggest δ15N and δ34S data also suggest that the only species that the range of values of organic material present in that are likely to be obtaining significant dietary car- the shallow sediments directly below the tube worm bon and sulfur directly from the symbiont-containing aggregations are –30 to –28‰ C, 0 to 5‰ N and –14 to bivalve species are the starfish Sclerasterias tanneri –2‰ S (Table 2). In addition to providing insights into and the Amphinomid polychaete (Table 2). Micheli et the biology of some of the individual species present at al. (2002) found that hydrothermal vent mussels were the seeps, this study clearly demonstrates the power of also palatable to some invertebrate predators, particu- using a third isotope (S) for trophic analysis of seep, larly crabs. Munidopsis sp. and an Atelecylidid crab in and presumably hydrothermal vent, communities. this study had high tissue δ15N values, reflecting their Since the seep communities were discovered in 1985 relatively high trophic level, however, their elevated (Kennicutt et al. 1985, Brooks et al. 1987) photographs δ34S relative to tube worms, and high δ13C relative to and observations have indicated that some fauna were mussels, indicate that neither of these chemoau- consistently associated with the seeps. The elevated totrophic-containing invertebrates was the dominant biomass and presence suggested that the closely asso- food item for these predators. This is further supported ciated fauna would derive the bulk of their nutrition by Bergquist et al. (2003) who note that among the from local chemoautolithotrophic production, even thousands of hydrocarbon seep tube worms examined though these communities are present in relatively in their study, none showed any signs of predation shallow waters where input of photosynthetic material (unlike their observations in hydrothermal vent is likely to be significant. This study supports the Ridgeia piscesae communities). hypothesis that fauna tightly associated with tube Based on what is known of isotopic discrimination worm aggregations at cold seeps in the Gulf of Mexico between trophic levels, the feeding biology of some of obtains the bulk of its nutrition from local source of pri- the species analyzed, and the limited mobility of those mary production. This study is consistent with previous 58 Mar Ecol Prog Ser 292: 51–60, 2005

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Editorial responsibility: Otto Kinne (Editor-in-Chief), Submitted: April 6, 2004; Accepted: February 17, 2005 Oldendorf/Luhe, Germany Proofs received from author(s): May 2, 2005