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Keywords:

  • Deep-sea symbiosis;
  • instantaneous fecundity;
  • oophagous bivalve;
  • vestimentiferan tubeworm

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The deep-sea bivalve Acesta oophaga lives attached to the anterior end of the vestimentiferan tubeworm, Lamellibrachia luymesi, at cold methane seeps. The bivalve is found almost exclusively on female tubeworms, where it consumes the lipid-rich eggs of L. luymesi that are spawned year round (Biological Bulletin, 209, 2005, 87). It is apparent that A. oophaga benefits directly from this close association, but the consequences for the tubeworm host may be more complicated than just a simple predator–prey interaction. Since A. oophaga completely surrounds the tube opening and plume of the worm, it is likely that its presence would limit oxygen uptake by L. luymesi, thereby inhibiting worm growth and reproduction. We hypothesized that occupied tubeworms would compensate for this by growing larger plumes for oxygen uptake. To explore the effects of bivalve presence/absence on female tubeworms, several morphological features, including body size, plume length, tube diameter, and tube segment length, as well as instantaneous fecundity, were compared. Results suggest that the mere presence of A. oophaga has a significant impact on the morphology of its host worm, as all measures of worm size, except for tube segment length, were significantly greater with clams present. Additionally, instantaneous fecundity was 3.5 times higher in occupied worms, implying that tubeworms are not oxygen-deprived or energy limited as a result of bivalve presence. Our findings suggest that the association between these two deep-sea organisms may be a more complex form of symbiosis than the simple predator–prey relationship, as previously thought.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Deep-sea vestimentiferan tubeworm aggregations provide rare hard substrata for organisms that would not otherwise be present at soft-bottom methane cold seeps (Carney 1994; Bergquist et al. 2003; Cordes et al. 2005). These close associations offer the opportunity to adapt to surrounding organisms, ultimately leading to symbiotic species with modified or specialized characteristics (Thompson 1999).

Among those organisms that have undergone such adaptive changes is the file-shell bivalve Acesta oophaga Järnegren 2007, which attaches itself to the anterior end of the tubeworm Lamellibrachia luymesi van der Land and Norrevang 1975 at cold methane seeps in the Gulf of Mexico (Fig. 1). The shell of this bivalve (often longer than 10 cm) is specifically evolved for holding the tube opening and plume of the worm inside its inhalant mantle cavity (Kohl & Vokes 1994; Järnegren et al. 2005). Lamellibrachia luymesi is found in dense aggregations at hydrocarbon seeps at depths <1000 m on the Louisiana slope of the Gulf of Mexico (Brooks et al. 1987; MacDonald et al. 1989). Like all vestimentiferans, this species lacks a gut, mouth, and anus, instead getting its nutrition from endosymbiotic chemosynthetic bacteria (Cavanaugh et al. 1981). Lamellibrachia luymesi produces large quantities of lipid-rich eggs year round, which are released from distally located ovisacs (Young et al. 1996; Hilario et al. 2005). These tubeworms grow very slowly, adding tube segments at the anterior end and reaching heights of over 2 m and ages of up to 200 years (Fisher et al. 1997; Bergquist et al. 2000).

image

Figure 1. A aggregation of Lamellibrachia luymesi with and without Acesta oophaga attached. Photograph was taken from Johnson-Sea Link II submersible near Brine Pool NR-1 cold seep at 750 m depth on the Louisiana Slope.

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Acesta oophaga juveniles settle near the base of the tubeworms and crawl upwards using a well developed foot and byssal threads (Järnegren et al. 2005). When juveniles reach the aperture of the tube, they begin to grow the characteristic notch of the adult shell, a modification that allows this species to completely surround the top of the tubeworm (Kohl & Vokes 1994; Järnegren et al. 2005). After becoming permanently attached, the clams shift from filter feeding to a predominantly oophagous existence (Järnegren et al. 2005). The bivalve, which preferentially lives on female worms, receives up to 72% of its metabolic requirements from L. luymesi eggs released into its mantle cavity (Järnegren et al. 2005). Moreover, the continuous supply of tubeworm eggs is sufficient to support year-round reproduction in the bivalve (Järnegren et al. 2007).

Although it is evident that the close association between A. oophaga and L. luymesi benefits the bivalve, it is unclear whether this interaction is more complex than the simple predator–prey relationship described by Järnegren et al. (2005). In this study, we explored the possibility that tubeworms might compensate for adverse effects of the association by increasing fecundity or by phenotypic changes in morphology. Specifically, we hypothesized that the presence of A. oophaga would limit oxygen uptake by L. luymesi, inhibiting growth and reproduction of the tubeworm. We also hypothesized that L. luymesi would attempt to compensate for this by increasing the size of its plume, which is used for oxygen uptake. We tested these hypotheses by examining several aspects of L. luymesi morphology and fitness, including body size, tube diameter, tube segment length and instantaneous fecundity, comparing individuals with and without the oophagous bivalve A. oophaga.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Lamellibrachia luymesi were collected on dives of the submersible Johnson Sea-Link II between 26 September and 4 October 2009 between depths of approximately 500 and 600 m at several sites on the Louisiana Slope: Brine Pool NR1 (27°43.4157′ N, 91°16.756′ W), Green Canyon GC-234 (27°44.7318′ N, 91°13.4355′ W) and Bush Hill (27°46.9478′ N, 91°30.5266′ W). Tubeworms with and without A oophaga were collected on each dive from the same aggregations. Specimens were brought to the surface in a closed plastic container on the submersible and transferred to the ship's cold room. Tubeworms and bivalves were held in 4–10 °C water and used within 3 days of collection. Acesta oophaga and their corresponding tubeworms were marked and kept together until measured. Vernier calipers were used to take linear measurements to a 10th of a millimeter.

Of the L. luymesi with A. oophaga attached, 95.7% were female (n = 24), whereas 48.6% (n = 72) of tubeworms without A. oophaga were female. Worm tubes were measured externally for a variety of characteristics and relationships between size variables were analyzed using Pearson correlations. Since tube growth occurs at the distal end where bivalves attach, lengths of the top 10 segments of each tube were taken for tube growth comparisons (Fig. 2). In addition, starting at the tube opening, the diameter of the tube was measured every 10 cm until the tube consistently reached a width of 4.5 mm. Since the part of the tube below the sediment was difficult to collect in its entirety, tube lengths were measured down to a diameter of 4.5 mm so that comparisons could be made between all tubes and worms collected. All tubes with bivalves attached contained a worm. However, some tubes without bivalves no longer held worms and were therefore not included in the analyses.

image

Figure 2. Measurements of external tube characteristics. SL, segment length; D, top tube diameter.

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Worms were extracted from their tubes by hydrostatic pressure from a syringe applied to the severed proximal end, then measured for plume length, vestimentum length and vestimentum width. Sex was determined by an obvious sexual dimorphism on the vestimentum region (Hilario et al. 2005) and the presence or absence of eggs in females (n = 44) was recorded. Instantaneous fecundity was measured by dissecting out the ovisacs, removing the oocytes under a dissecting microscope and suspending them in a known volume of filtered seawater from which three subsamples were counted and averaged. Acesta oophaga shell length and maximum shell width (at right angle to the length) were also measured.

Because of differences in plume and segment lengths between male and female L. luymesi (see 'Results'), and since most worms with bivalves attached were females, only characteristics of female worms were compared to explore the effects of A. oophaga on tubeworms. Therefore, any differences in worms or their tubes could be attributed to bivalve presence and not to the sex of L. luymesi. Two-sample t-tests were performed to compare differences between male and female worms, as well as size and fecundity differences for females with and without bivalves present.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The relationship between Lamellibrachia luymesi tube size and worm body size was investigated to compare males and females with no Acesta oophaga attached. Neither vestimentum length nor plume length was correlated with tube length, and vestimentum length was not correlated with tube diameter (Pearson correlation coefficient, r < 0.2, P > 0.3). However, females were found to have significantly longer plumes (mean = 8.62 mm) than males (7.68 mm) (two-sample t-test, t70 = –2.08, P = 0.042). Plume length was weakly correlated with tube diameter for both males (Pearson correlation coefficient, r = 0.52, P = 0.012) and females (Pearson correlation coefficient, r = 0.67, P = 0.018). There was also a trend, although not statistically significant, indicating that females (mean = 3.50 mm) had longer tube segments than males (3.03 mm) (two-sample t-test, t70 = –1.67, P = 0.099).

Significant differences in female worm sizes were found between those with A. oophaga present and those without. Vestimentum length (two-sample t-test, t29 = 2.52, P = 0.015), width (two-sample t-test, t29 = 2.85, P = 0.006), and area (two-sample t-test, t29 = 2.80, P = 0.009; Fig. 3A) were all significantly greater for tubeworms with clams (clams present mean length = 50.0 mm, absent = 43.0 mm; present mean width = 16.09 mm, absent = 13.82 mm; present mean area = 800 mm2, absent = 598 mm2). In addition, vestimentum area had a positive and statistically significant correlation with bivalve shell width (Pearson correlation coefficient, r = 0.69, P = 0.001; Fig. 4A). Mean plume length was also significantly greater (two-sample t-test, t42 = 2.43, P = 0.019) in L. luymesi on which A. oophaga were present (present = 10.08 mm, absent = 8.62 mm; Fig. 3B). In addition, the length of the plume was positively and significantly correlated with the size (shell width) of the clam present (Pearson correlation coefficient, r = 0.73, P < 0.001; Fig. 4B).

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Figure 3. Comparison of four major morphological characteristics between Lamellibrachia luymesi with and without Acesta oophaga attached. All four graphs show mean dimension in millimeters (±SE). Vestimentum area (A), Plume length (B), and top tube diameter (D) were all significantly greater in tubeworms with bivalves attached. Segment length (C) was significantly shorter for worms with clams present.

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image

Figure 4. Correlations between bivalve size and host worm size. Vestimentum area (A), plume area (B), and top tube diameter (C) of worms show a positive and significant relationship with bivalve width (mm).

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Differences in tube characteristics between female worms with and without bivalves present were also evident. The average length of the top 10 segments of L. luymesi tubes was significantly shorter in tubeworms with A. oophaga present (present mean = 1.95 mm, absent = 3.50 mm) (two-sample t-test, t55 = –5.08, P < 0.001; Fig. 3C). Tubeworms with A. oophaga attached also had significantly wider top tube diameters (mean = 9.62 mm) (two-sample t-test, t43 = 3.22, P = 0.002) compared with those without clams (mean = 8.57 mm; Fig. 3D). The top tube diameter had a positive and significant correlation with A. oophaga shell width (Pearson correlation coefficient, r = 0.70, P < 0.001; Fig. 4C), whereas there was no significant correlation between top segment lengths and clam shell width (Pearson correlation coefficient, r = 0.04, P = 0.83). Total tube length (measured down to a tube diameter of 4.5 mm) was significantly larger for worms with bivalves present than absent (present mean: 100.7, absent: 69.0 cm) (two-sample t-test, t23 = 3.34, P = 0.003).

Instantaneous fecundity differed significantly as well; female tubeworms with A. oophaga had almost four times more eggs (two-sample t-test, t16 = 2.41, p = 0.028) than females without A. oophaga (present mean = 23 774 eggs, absent mean = 6353 eggs; Fig. 5). Top tube diameter was not correlated with the instantaneous fecundity of the tubeworm (Pearson correlation coefficient, r = –0.08, P = 0.73), nor were tube segment length, vestimentum length or width, plume length, or size of A. oophaga (all P > 0.4). There was also no correlation between tube length and any measure of internal or external worm size (all P > 0.4). Approximately 77% (n = 22) of female tubeworms with A. oophaga had eggs at the time of sampling, whereas only 60.8% (n = 22) of tubeworms without A. oophaga had eggs at that time. There was no significant correlation between A. oophaga size and worm fecundity (Pearson correlation coefficient, r = –0.46, P = 0.08).

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Figure 5. Mean instantaneous fecundity of tubeworms (number of eggs × 1000) with and without Acesta oophaga attached (±SE). Worms with bivalves attached had almost four times greater fecundity than worms with no bivalve present.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The disproportionately high number of Acesta oophaga found on female Lamellibrachia luymesi (95.7%) is consistent with the findings of Järnegren et al. (2005) and strongly supports the conclusion that A. oophaga select female L. luymesi for the purpose of feeding on their eggs. We hypothesized that the heavily vascularized plumes of L. luymesi might grow larger in occupied tubeworms because of competition for oxygen with the ciliated gills of the clams, as the gas exchange systems of both species lie in close proximity within a restricted mantle cavity. This hypothesis was supported by the data. Plume length was significantly longer in tubeworms with bivalves. However, since the occupied worms invested more energy in plume size, it was somewhat surprising that they also were able to invest additional resources in fecundity and in tube length. A filter-feeding bivalve could supply L. luymesi with nitrogen from its waste products or dissolved organic matter. The potential role of the bivalves in supplying carbon, nitrogen and other nutrients to the symbiotic chemoautotrophic sulfide-oxidizing bacteria in the trophosome of L. luymesi (Cavanaugh et al. 1981; Freytag et al. 2001; Dattagupta et al. 2006) would be an interesting topic for future research. An energy budget for the entire symbiosis (worm, clam, and bacteria) is needed. Any increase in the production of the trophosome bacteria would enhance the energy supply of the tubeworm. This, coupled with less energy being put towards vertical tube growth, might free up a substantial amount of energy that could be allocated to fecundity and growth.

Since there is no measure of worm size correlated with worm fecundity, the observed patterns of enhanced fecundity cannot be attributed to worm age or to selection of larger worms by the bivalves. It is more parsimonious to suggest a causal relationship in which the bivalve stimulates increased investment in growth and egg production. Moreover, bivalve size was correlated positively with tubeworm plume length, vestimentum area, and top tube diameter. Since larger clams are probably older than small ones and there is no evidence that bivalves change hosts during their later life, bivalve size can be used as a proxy for length of time spent on an individual tubeworm. Thus, it is reasonable to expect that morphological changes would be greater in L. luymesi that have hosted bivalves for longer periods of time (larger clams). It is also possible that differences between worms with and without bivalves are due to differences in worm age. If tube length is used as a proxy for age, the longer tubes found in worms with A. oophaga attached may suggest that they are older than those with no bivalves. However, since no correlation was found between tube length and segment length, top tube diameter, plume length, vestimentum area or fecundity, ‘age’ does not explain any of the patterns shown in this study. While the A. oophaga host selection process could be influenced by tubeworm age, the lack of a correlation between tube length and other morphological characteristics indicates that age is unlikely to be causing the patterns observed in this study.

Our results suggest that even simply the presence of A. oophaga, irrespective of clam size (or length of time present), has a significant impact on the morphology of their host worm. All measures of worm size (vestimentum width and length, plume length, and top tube diameter) were significantly greater with clams present, except for the length of the top 10 segments, which was reduced (Fig. 3). Thus, while worm size was enhanced by clam presence, shorter but wider top segments suggest an overall shift from vertical tube growth to horizontal tube growth (Fig. 3C and D). It is possible that the weight of the bivalve could inhibit vertical growth of the tubeworm, stunting its segments or forcing the worm to expend energy thickening or widening the tube. Whereas extra weight could influence tubeworm morphology, it is unlikely to have a direct effect since A. oophaga size was not correlated with segment length and the clam is not attached directly on top of the growing portion of the tube, but on the side. Presumably, vertical growth is normally a way for L. luymesi to enhance oxygen uptake by reaching areas of higher water flow. Perhaps the presence of a clam continuously filtering water over its gills would also increase the flow over the plume of its host worm, making vertical growth for oxygen capture no longer necessary for that individual tubeworm. In this case, a longer plume would be more beneficial for oxygen uptake than an increase in vertical height, which would be ineffectual for a worm hosting a bivalve.

An increase in energy, due to supplemental oxygen or nutrients, or a reallocation of energy from vertical growth to reproduction could explain the four times higher instantaneous fecundity found in female tubeworms with A. oophaga present than in those without. Similarly, more tubeworms were ovigerous when clams were present than absent (77 versus 60.8%), suggesting that more energy could be put towards egg production in worms hosting clams. While it is possible that A. oophaga could be choosing or moving to L. luymesi that have higher fecundities, this seems unlikely since the morphology of each bivalve is specific to its tubeworm host and their sizes are correlated, suggesting it has been present from either a young age or a significant amount of time. Since the instantaneous fecundity of L. luymesi was not found to be significantly associated with any measure of tubeworm size, internal or external, A. oophaga must be choosing female tubeworms based on a chemosensory mechanism. However, the small number (4%) of A. oophaga found attached to males instead of females indicates that a chemosensory mechanism, if present, may not be finely tuned enough to detect differences in fecundity. Therefore, any differences in fecundities are likely due to the presence, not the preference, of the clam.

The observed increased fecundities in worms with A. oophaga could also simply be due to the worms holding onto their eggs longer before release. Fertilized eggs were observed in L. luymesi that had bivalves attached, suggesting that the presence of the clam does not inhibit fertilization success. Since A. oophaga is known to consume the tubeworm eggs (Järnegren et al. 2005), it is likely that having more eggs to release at one time, could ensure that some of them escape by exceeding the clearance rate and effectively ‘swamping’ the bivalve predator. Similarly, a wider top tube diameter may allow for more eggs to escape. Thus, reproductive success in an occupied tubeworm could be enhanced either by an actual increase in tubeworm fecundity or by a change in spawning behavior. We do not know how often tubeworms spawn either with or without bivalves present.

Järnegren et al. (2005) classified the interaction of A. oophaga with its tubeworm host as one of simple egg-predation because there was no evidence that the host itself was harmed, as would be the case in parasitism. Our data support the lack of parasitism since we saw no apparent adverse effects on female worm growth or fecundity. However, our results also indicate that bivalves might have positive effects on the tubeworms. Lamellibrachia luymesi with A. oophaga attached showed increases in vestimentum length and width, plume length, tube diameter (Fig. 3), and instantaneous fecundity (Fig. 5). The tubeworms do not appear to be oxygen-deprived or energy-limited, and instead may be positively affected by the presence of the clam. However, although A. oophaga does not harm the adult worm directly and in fact seems to stimulate increased fecundity (a conventional measure of fitness), predation on the spawned oocytes must certainly reduce the tubeworm's contribution of propagules to future generations. Our results hint at a more complex form of symbiosis between these two deep-sea organisms rather than a mere egg-predator relationship. Further investigation of energy budgets, fecundity, spawning frequency, and the percentage of eggs consumed by A. oophaga could reveal that the relationship is a unique form of mutualism.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This study was supported by the National Science Foundation grant OCE 0527139 to C. M. Young, R. B. Emlet, and A. M. Wood. Zair Burris and Joshua Lord were supported by the NSF Graduate Teaching Fellows GK-12 grant DGE-0638731.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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