Polar lipid fatty acids as indicators of trophic associations in a deep-sea vent system community


Ana Colaço, IMAR – Department of Oceanography and Fisheries University of the Azores, Cais de Sta. Cruz, PT- 9901-862 Horta (Azores), Portugal.
E-mail: acolaco@notes.horta.uac.pt


The polar lipid fatty acid (PLFA) profiles of invertebrates living in chemosynthetic communities can indicate the degree to which these animals depend on specific types of bacteria. To identify the nutritional sources of various species from deep-sea hydrothermal vents of the Mid-Atlantic Ridge, a Principal Component Analysis was performed using individual PLFA profiles as descriptors. Two associations representing different feeding groups were identified: (i) mussels, commensal polychaetes and gastropods, (ii) shrimps and crabs. The first association relies more on sulphide-oxidizing bacteria, while the second one has more anaerobic sulphate-reducing bacteria biomarkers. Other small invertebrates reveal different diets. The polychaete Amathys lutzi shows the most diversified bacterial diet, with fatty acid biomarkers from both S-oxidizing and S-reducing bacteria.


It is widely known that fatty acid (FA) analyses can be good indicators of specific microorganisms, as different groups of bacteria may have different FA compositions (Lechevalier 1977; Goodfellow & Minnikin 1985). Hence, FAs have been used in classical enumeration procedures to reveal and estimate which microorganisms are associated with biofilms, soils and sediments (Guezennec et al. 1998) (Table 1).

Table 1.   Most abundant fatty acids biomarkers for the stated microorganisms.
ArchaeaGlycerol EtherGuezennec (1995)
PhytoplanktonC20:5ω3; C20:6ω3Chuecas & Riley (1969)
Desulfovibrio (Sulphate reducer)iC17:1ω7c; iC15:1ω7c; iC19:1ω7c;Nichols et al. (1986)
Desulfobacter (Sulphate reducer)10Me16; cyC18Boon et al. (1977)
ThiooxidizingC16:1ω7; C18:1ω7McCaffrey et al. (1989)
MethanotrophsC16:1ω5t; C16:1ω6; C16:1ω8; C18:1ω6; C18:1ω8Nichols et al. (1987); Bowman et al. (1993)
Thiobacillus sp. (Sulphur oxidizer)iC17:1ω510-11 Me C18:1ω6;Kerger et al. (1986)

Polar lipid fatty acids (PLFA) are good biomarkers for disclosing and describing trophic relations between vent community animals, as well as for confirming the importance of bacteria in the nutrition of organisms endemic to hydrothermal vents. Highly unsaturated fatty acids (HUFA) composed of long carbon chains (e.g. C20:4ω6, C20:5ω3 or C22:6ω3) can be indicative of phytoplankton (Chuecas & Riley 1969), although some bacteria can also synthesize them (Nichols 2003). These HUFA are characteristic of all heterotrophic marine invertebrates and are usually present in high concentrations in the tissue of these organisms (Sargent et al. 1990). Therefore, very low levels of HUFA in the hydrothermal vent communities suggest a trophic web that is not primarily based on phytoplankton. Likewise PLFA in invertebrates living in communities where the food chain is based on microorganisms can indicate the degree to which these animals feed on bacteria and suggest which bacteria they rely on, as bacteria-specific PLFA can be used as biomarkers (Guezennec 1995).

It has been proposed that animals whose diets predominantly consist of bacteria, i.e. diets rich in 16:0, 16:1(n-7), and 18:1(n-7) FAs, while being relatively deficient in (n-3) polyunsaturated fatty acids (PUFAs), produce Non-methylene interrupted dienoic (NMID) FAs from monenoic FAs (Ackman & Hooper 1973), which effectively substitute for the low levels of (n-3) PUFAs (Pond et al. 2002). This FA has been observed in symbiotic organisms and some molluscs (Conway & McDowell Capuzzo 1991).

Several authors have studied FA composition and compound-specific stable isotopes of Mid-Atlantic Ridge hydrothermal vent invertebrate species (Pond et al. 1997a,b, 1998; Rieley et al. 1999; Allen et al. 2001). Although these studies have contributed greatly to the knowledge of the feeding strategies of individual species, they did not establish trophic links between species and their respective predators. A special case is the dominant vent shrimp at the deeper Mid-Atlantic Ridge hydrothermal vent sites, Rimicaris exoculata. It is the only species that actively seeks the active facies from the hydrothermal structures and several hypotheses developed the importance of bacteria in the nutrition of these shrimps. In this study we examined: (1) What is the relationship between the lipid profiles of the various vent species? (2) Can trophic relationships be established from PLFA profiles? (3) What are the feeding strategies of R. exoculata?

Material and Methods

Animal collection

Samples were taken during three deep-sea hydrothermal vent cruises along the Mid-Atlantic Ridge (MAR). MAR/97, in 1997, sampled all of the known hydrothermal vent fields along the MAR at that time. (Menez Gwen 37.51° N 840 m; Lucky Strike 37.18° N 1700 m; Rainbow 36.13° N 2300 m; Broken Spur 29.10° N 3000 m; TAG 26.08° N 3600 m; Snake Pit 23.22° N 3400 m, and Logatchev 14.45° N 2900 m). Two other cruises (MARVEL in 1997 & PICO in 1998) sampled the Azores Triple Junction hydrothermal vent fields (Menez Gwen; Lucky Strike; Rainbow). Animals were collected using the submersible's packman scoop, or by using a slurp gun. Mirocaris fortunata, Chorocaris chacei, and Rimicaris exoculata (shrimps); Bathymodiolus spp. (mussels); Segonzacia mesatlantica (crab); and Phymorhynchus moskalei (whelk) were dissected immediately upon arrival on board to retrieve the digestive gland. The mouth parts were retrieved only from the R. exoculata shrimp. The remaining specimens collected were not dissected due to their reduced size. Samples were subsequently deep-frozen (−80 °C) on board and freeze-dried at the shore laboratory. Whole animal PLFA profiles were determined in the polychaete Amathys lutzi (n = 5), the polychaete Branchipolynoe seepensis (n = 12) commensal with mussels, the pycnogonid Sericosura sp. (n = 2), the amphipods (n = 2) (unknown species) and the shrimp Alvinocaris markensis (n = 1).

Polar lipid fatty acid profiles were also determined for the digestive glands of the mussels (n = 26) (Bathymodiolus azoricus, B. puteoserpentis and Bathymodiolus spp.), crab (n = 15) (S. mesatlantica), whelk (n = 6) (Phymorhynchus sp.), and shrimps (R. exoculata (n = 13), C. chacei (n = 2) and M. fortunata (n = 6)), along with R. exoculata mouthparts (n = 4).

Lipid analysis

Lipids were extracted using a modified version of the Bligh and Dyer method (Bligh & Dyer 1959; White et al. 1979). Total extractable lipids were fractionated by silicic acid column chromatography. The PLFA esters were methylated by acid methanolysis of the polar lipid fractions. Quantification was based on comparing peak areas to an internal injection standard (19:0). Gas chromatography was carried out using a HRGC 5160 Megaseries (Carlo-Elba) equipped with an injection system on column, and with a flame ionization detector, connected with an integrator SP42070 (Spectra Physics) D-2500 (Merck). Chromatography was carried out using a non-polar column CP-Sil-5 CB® (Chrompack).

FA structural verification

Two-derivatization methods (DMDS and D-MOX) were used to identify the mono and polyunsaturated double bond position (Guezennec 1986; Nichols et al. 1986; Yu et al. 1989; Fay & Richli 1991). FA identification was by GC/MS. GC-MS was carried out using a HRGC 5160 Megaseries (Carlo-Elba) chromatograph equipped with a NERMAG R 10 × 10 quadripolar mass spectrometer.


To identify associations between the different species, principal component analysis (PCA) was performed using individual PLFA profiles as descriptors for the different individuals. The matrix was standardized by dividing each row by the standard deviation and subtracting off the mean of each row.

We hypothesized that if the species fall within the same group, it signifies a strong association among the species. They either eat the same food source or feed on each other. The higher trophic level (predation or omnivory) is established when two species are associated and one presents lower amounts of bacterial origin FA.

As a result of the low number of samples, no statistical analysis was performed to compare the PLFA profiles of the digestive glands and mouth parts of the shrimp R. exoculata. Graphs are presented comparing the PLFA profiles.


Relationships between taxonomic groups

Table 2 summarizes the percentage of each PLFA for each species or taxonomic group. Several PLFA profiles were assessed for each taxonomic group; however, no specific PLFA profile could be established for a species or taxonomic group, although the percentages obtained in some cases merit attention.

Table 2.   Fatty acid composition of polar lipids expressed as a percentage of the total fatty acids for each taxonomic group.
fatty acidsAmathys lutzi (all) LS = 2; Rb = 2Branchipolynoe seepensis (all) LS = 8; Rb = 1; SP = 1Bathymodiolus spp. (dg) MG = 4; LS = 14; Rb = 4; BS = 1; SP = 2; Lg = 1Rimicaris exoculata (dg) LS 1; Rb = 6; BS = 1; SP = 2; TAG = 2; Lg = 1Mirocaris fortunata (dg) LS = 4; Rb = 2Chorocaris chacei (dg) LS = 1; BS = 1Sericosura sp. (all) LS = 2Amphipod (all) MG = 1; LS = 1Alvinocaris markensis (dg) SP = 1Phymorhynchus sp (dg) LS = 2; Rb = 2; SP = 1; TAG = 1Segonzacia mesatlantica (dg) LS = 6; Rb = 3; BS = 2; TAG = 2; Lg = 2
  1. MG = Menez Gwen; LS = Lucky Strike; Rb = Rainbow; BS = Broken Spur; SP = Snake Pit; Lg = Logatchev and are indicative of the number of animals studied (not the number of profiles) for each vent field. dg  = digestive gland. On the fatty acids; Br = branched.

Unknown a0000.210000.71000
Unknown b003.0000000000
Unknown c000.1300000000

The pycnogonid Sericosura sp. and the Mirocaris fortunata and C. chacei shrimps had a high percentage of C18:0. For mussels and the polychaete Amathys lutzi, C16:0 was present at more than 20%. The shrimps Rimicaris exoculata and M. fortunata had large amounts of C14:0.

The percentage of monounsaturated fatty acid (MUFA) varied considerably among all the groups. The shrimp R. exoculata was the only species that possessed C14:1ω7. The FAs C16:1ω9 and C16:1ω7 were present at levels of more than 8% in all the groups except for the pycnogonid Sericosura sp. and the whelk Phymorhynchus sp. The highest percentages of C18:1ω13 + C18:1ω9 were found in the amphipods, the shrimp A. markensis, and the crab S. mesatlantica. C18:1ω7 was important in all taxonomic groups except for the mussels. The whelk Phymorhynchus sp. had noticeable amounts of C20:1ω13 and C20:1ω9 as did the commensal worm (B. seepensis) and the mussels. C20:1ω7 was present in similar amounts in the polychaete A. lutzi, commensal worm, mussel and whelk, but was present at low levels in the other taxonomic groups. C21:1ω9 was found exclusively in the polychaete A. lutzi.

The highest percentages of branched PLFA were seen in the polychaete A. lutzi and the shrimps R. exoculata and M. fortunata. However, iC19:0 was an exception, with the highest percentages found in the mussel, followed by the shrimp M. fortunata and the amphipod. PUFAs were present in different quantities in all the taxonomic groups sampled. Nearly all the animals contained NMID fatty acids from the Δ5-desaturation pathway. A higher percentage of the PUFA C18:3ω (9,12,15) was present in mussels and amphipods than in the other taxonomic groups (<1%). High proportions of the NMID C20:2ω(7,15) were found in mussels, the shrimp R. exoculata and the whelk Phymorhynchus sp. The mussel and commensal worm had relatively high percentages of the NMID C20:2ω(6,15), as did the whelk and the amphipods. The percentage of NMID C20:2ω(8,15) was greater than 1% in the polychaete A. lutzi, in the commensal worm, mussels, whelk, amphipod and in the shrimp M. fortunata.

The PLFA profiles of the digestive gland or the entire animal were used as descriptors to perform the PCA (Fig. 1). The first component differentiates the mussels Bathymodiolus spp., the Phymorhynchus sp. whelk and the commensal worm B. seepensis. The shrimps and crab are grouped separately, and at the center of the axes other non-dominant invertebrates, such as the polychaete A. lutzi, the pycnogonid Sericosura sp., and amphipod, are identified. The second component differentiates the mussel, shrimps and part of the crab from the commensal worm, the whelk Phymorhynchus sp., the polychaete A. lutzi and the remaining crabs.

Figure 1.

 Principal component analysis performed on the fatty acid profiles of all samples. Component 1 represents 16.81% of the variance, and component 2 represents 12.52% of the variance. The eigenvalues are 14.80 and 11.04.

Particular cases

Shrimp Rimicaris exoculata

For a better understanding of the R. exoculata food source, the PLFA profiles of mouthparts and digestive gland were compared (Fig. 2). The profiles of these tissues in R. exoculata individuals, three from Rainbow hydrothermal field and the other from Lucky Strike hydrothermal field, overlap. With the exception of some NMID, both individuals exhibited similar PLFA profiles. Generally the mouthparts presented higher quantities of the same MUFAs and branched FAs than the digestive gland.

Figure 2.

 Polar lipid fatty acid profiles from Rimicaris exoculata mouthpart and digestive gland. A: Rainbow hydrothermal vent field (n = 3). B: Lucky Strike hydrothermal vent field (n = 1).

Mussels (Bathymodiolus azoricus, B. puteoserpentis, Bathymodiolusspp.)

Despite the existence of S-oxidizing and methanotrophic endosymbiotic bacteria in mussel gill tissue (Fiala-Médioni et al. 2002), no typical methanotrophic bacteria biomarkers were found. However, the large percentage of iC19:0 and C18:1ω13 + C18:1ω9 is certainly related to the presence of endosymbiotic bacteria.


The presence of bacteria-specific biomarkers such as iC15:0 and iC17:1ω7, as well as large quantities of the 1ω7 series, low levels of PUFA, the absence of HUFA (notably the absence of C20:5ω-3 and C22:6ω-3, which are phytoplankton biomarkers) and the presence of NMID all indicate that the vent communities rely almost entirely on bacteria. Those species that present more branched FAs from the ω7 series rely more on S-reducing bacteria (SRB), while the ones with more MUFA from the ω7 series and more NMID from this series rely more on S-oxidizing bacteria.

Relationships between species

The most striking result of the PCA analyses was the observation of two different groups. Despite the fact that no species presented a specific PLFA profile, organism associations were found. The two associations: mussel–commensal worm–whelk and shrimps–crab have different PLFA indicative of different bacteria in their diets. The mussel–commensal worm–whelk association relies more on S-oxidizing bacteria, while the crab–shrimp association has more SRB anaerobic biomarkers. This indicates that the latter group of animals apparently lives in conditions closer to anoxia than the other, which is congruent with the microdistribution of these organisms (Desbruyères et al. 2001). The large central group represents omnivorous species that feed on bacteria in addition to other types of food (e.g. C. chacei). The individual FAs do not differentiate groups but the percentage and composition of PUFA are responsible for the observed distribution.

These two associations (mussel–commensal worm–whelk and shrimps–crab) were also demonstrated by stable isotope analysis, with the first association presenting very depleted δ13C (around −30‰), and the second presenting less depleted δ13C (around −11‰) values (Colaço et al. 2002).

The individuals at the center of the PCA axes have a more diversified bacterial diet, with several different biomarkers from S-oxidizing and S-reducing bacteria. The association of mussels and Phymorhynchus sp. was also observed through stable isotope analysis (Fisher et al. 1994; Colaço et al. 2002) as was the mussel–commensal worm coupling (Colaço et al. 2002).

The FA composition of M. fortunata fell midway between that of the other shrimp species (R. exoculata, C. chacei, A. markensis). Rimicaris exoculata is considered to be bactivorous, and C. chacei and A. markensis to be carnivorous or scavengers. The PLFA profile of M. fortunata indicates that this species is omnivorous, feeding not only on other animals (carnivorous) but also on S-oxidizing bacteria (bactivorous), as indicated by the presence of branched FAs, monounsaturated ω7 FAs and more NMID (cf. Table 2). However, according to our definition, this species might also be a secondary consumer, eating animals that are eating the bacteria, as was indicated by Colaço et al. (2002). The crab S. mesatlantica shows a similar pattern to that of M. fortunata, despite possessing fewer branched FAs. According to Colaço et al. (2002) the crab's trophic position is not that of omnivore, but rather predator, feeding on bactivorous animals like amphipods and other mixotrophs like shrimps. If this is the case, the bacterial FA present in these animals is from the prey food source and not from bacteriophagy.

The polychaete A. lutzi presented a characteristic FA profile, with the higher amounts of branched FAs indicative of a diversified bacterial diet. As a non-selective deposit feeder, it can eat different particles from the hydrothermal vent environment.

Detailed analyses of the FA profiles show an absence of HUFA (C20:5ω3 and C22:6ω3) in all the taxonomic groups studied, suggesting that if the vent fauna relies on photosynthetically produced material, it is as transformed material like nutrients or polysaccharide molecules, rather than lipids. The low amounts of PUFA also support this idea, especially in light of the absence of C20:4ω6.

The presence of phyto-biomarkers at some hydrothermal vent fields (e.g. 13° N) and their absence at the Galapagos field were tentatively related to the mussel condition. The condition parameter was the loss of symbionts (Ben-Mlih et al. 1992). This situation is not surprising, as the mussels did not lose their filter-feeding capacity, and in the absence of symbionts, phytodetritus-derived particles would be one of the available energy sources. Mussel symbionts disappear when the mussel environment loses the symbionts’ energy source (P. Dando, personal communication).

The PUFA present in the PLFA lipids of the studied species are due to two factors, the desaturation possible in all aerobic Δ9 organisms, and the desaturation specific to certain Δ5 marine invertebrates. The presence of high proportions of NMID from C18:1ω7 is a clear indicator that the species rely on chemoautotrophic bacteria, as has already been indicated by several authors (Zhukova 1991; Pond et al. 1997b; Pranal et al. 1997).

Particular cases

Shrimps Rimicaris exoculata

In the deep-sea hydrothermal vent fields at MAR, the three main sources of dietary carbon that are available to the shrimp R. exoculata are: (i) bacteria fixed on the mouthparts of this species; (ii) bacteria associated with the minerals that these organisms ingest from the sulphide chimneys; and (iii) detritus from the oceanic photic zone. The absence of HUFA and the low levels of PUFA with the exception of the NMID, make the third hypothesis rather improbable, consistent with the results of Rieley et al. (1999). The lipid profile of this species is also different from that of deep-sea benthic shrimps (Nematocarcinus gracilis) that rely on phototrophically derived organic matter (Allen 1998). However, according to Pond et al. (1997a), juvenile R. exoculata rely on material from the photic zone of the ocean. Allen et al. (2001) also showed that in this species, adults and juveniles exhibit different partitioning of lipids in tissues, with an increased proportion of bacterial-derived organic matter in adults compared with that of juveniles.

Comparison of mouthparts and digestive gland PLFA profiles revealed a close similarity, with the digestive gland having more NMID. When looking at the MUFAs that might underlie the origin of these NMID, the FA profiles of the mouthpart and the digestive gland are very similar. Earlier studies showed that the bacteria from the mouthparts (epibionts) and bacteria from sulphides are closely related (Rieley et al. 1999). However, by means of a specific compound stable isotope analysis, it was shown that the epibiont bacteria have a δ13C signal similar to that of the shrimp, while the sulphide bacteria are much more 13C depleted. Considering these factors, and from the results presented here, we conclude that this species relies on bacteria present in its mouthparts.

Mussels (Bathymodiolus azoricus, B. puteoserpentis, Bathymodiolus spp.)

Analyses of FAs as biomarkers often allow for an evaluation of host–symbiont energy relationships and carbon sources (Jahnke et al. 1995; Pranal et al. 1997).

Despite the fact that the mussels host S-oxidizing and methanotrophic bacteria (Fiala-Médioni et al. 2002), no methanotrophic biomarker was observed in the mussel PLFA profiles. This result contradicts Pond et al. (1998), in which these biomarkers were found in mussels from Lucky Strike and Menez Gwen. Type I methanotroph biomarkers (C16:1ω6; and C18:1ω6) were observed in other species but in smaller amounts. The presence of the FA C18:1ω13, a methylotrophic bacteria FA, does not allow the determination to which type of methanotrophic bacteria the mussels rely on, as methylotrophs are ubiquitous microorganisms that can use C1 compounds (methanol, methylamine, methane, etc.) for growth and only some specialized methylotrophs, are the methanotrophs. Bowman et al. (1991) characterized several methanotrophic bacteria from PLFA profiles and showed that some bacteria did not present the typical biomarkers of the methanotrophic types. Despite the lack of typical methanotrophic PLFA, the presence of other methanotrophic bacteria cannot be excluded. The presence of NMID C20:2ω6, 15 and C20:2ω8,15 in mussels leads us to believe that these FAs are the results of a specific biosynthetic pathway based on chain elongation and original Δ5 desaturation of the FAs C16:1ω6; C16:1ω8; 18:1ω6 and C18:1ω8 (which are methanotrophic biomarkers). The methanotrophic FA published in Pranal et al. 1997, were not detected in the mussels. As the deep-sea hydrothermal environment is continually changing, the hypothesis that the mussels have lost methanotroph symbionts and began using other symbionts cannot be ruled out. The presence of S-oxidizing bacteria biomarkers (C16:1ω7; C18:1ω7; and C20:11ω7) and NMID derived from FAs (e.g. C18:3ω7,10,13; C20:3ω7,10,15; C20:2ω7,15) and the absence of HUFA are consistent with the results of Pond et al. (1998) and with other studies on animals hosting S-oxidizing endosymbiotic bacteria or feeding on them (Conway & McDowell Capuzzo 1991; Zhukova 1991; Zhukova et al. 1992; Fullarton et al. 1995; Pranal et al. 1997). The presence of PLFA from SRB in mussels proves that they continue to be filter feeders.


Fatty acids from polar lipids can be used to reveal associations among invertebrate species, revealing carbon utilization patterns between species and identifying the organisms that rely on the same type of bacteria. Such associations imply that the species eat the same food types, or that there is a prey–predator relationship. The associations revealed here showed that some species rely more on S-oxidizing bacteria (mussel–commensal–whelk), while the shrimp and crabs rely more on SRB. The PLFAs can be used to establish trophic relationships in the vent environment. However to generalize, there is a need to validate the hypothesis that prey and predator present similar FAs or elongation, and this has only been done for the relationship between the primary producers (bacteria and phytoplankton) and the primary consumers.


We thank the Nautile and Alvin teams, the crews of the R.V. Atalante, Nadir, and Atlantis and chief scientists Anne-Marie Alayse, R. Vrijenhoek and R. Lutz. Financial support for this research was provided by MAST 3 AMORES-CT MAS3 950040 and the ‘Fundação para a Ciência e Tecnologia-Programa Praxis XXI (Portugal)’. We would like to thank the anonymous reviewers who improved the manuscript. This paper is a contribution to project ChEss of the CoML and to the FP6 NoE MarBEF (contract no. GOCE-CT-2003-505446).