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Keywords:

  • Bathymodiolus childressi;
  • Bathynerita naticoidea;
  • chemical cue;
  • deep sea;
  • Gulf of Mexico;
  • hydrocarbon seep

Abstract

  1. Top of page
  2. Abstract
  3. Problem
  4. Material and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References

Bathynerita naticoidea (Gastropoda: Neritidae) is a numerically dominant heterotrophic gastropod found at hydrocarbon seep sites on the upper Louisiana slope of the Gulf of Mexico. Snails of this species are commonly associated with beds of the methanotrophic mussel Bathymodiolus childressi (Bivalvia: Mytilidae), and their population structure mirrors that of the mussels they are found among. Previous studies have shown that these snails feed on bacteria and decomposing periostracum on the B. childressi shell. We predicted that B. naticoidea might be attracted to cues specific to its preferred habitat, such as dissolved methane, mucus from conspecific snails, or metabolites produced by B. childressi mussels. To examine this, we used a flow-through Y-maze system to investigate the behavior of B. naticoidea exposed to these potential cues. We found that the nerite is not attracted to methane, but is strongly attracted to seawater conditioned with B. childressi. The attractant appears to be specific to this type of mussel, and is not a soluble cue produced by conspecific snails.


Problem

  1. Top of page
  2. Abstract
  3. Problem
  4. Material and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References

Chemoreception is widely documented in shallow marine ecosystems (Zimmer & Butman 2000), where it is used by a variety of organisms for purposes such as food detection (Salierno et al. 2003), predator avoidance (Rahman et al. 2000), and coordination of larval release and settlement (Krug & Manzi 1999). Although chemical communication is likely to be pervasive in the deep sea (Herring 2003), it is poorly studied due to difficulties of working in this habitat. Deep-sea cold seep communities contain high densities of organisms (Sibuet & Olu 1998; Levin 2005), and exist as spatially isolated patches in a larger environment lacking sufficient reduced chemicals to support them (Brooks et al. 1987; Carney 1994). In these ecosystems, chemical cues may be crucial for colonization and persistence by the largely endemic fauna (Carney 1994).

The Gulf of Mexico seafloor has multiple locations at 400–3000 m depths where hydrocarbon seepage supports extensive populations of chemosynthetic organisms (Brooks et al. 1987; MacDonald et al. 1990a; MacDonald 1998). The mussel B. childressi, which derives most of its nutrition from methanotrophic endosymbionts in its gills (Childress et al. 1986; Cary et al. 1988; Streams et al. 1997), is a prominent foundation species of these communities between 500 and 2200 m depths. Beds of these mussels can be extensive (MacDonald et al. 1990b) and provide habitat for several species of heterotrophic fauna (Bergquist et al. 2005). The gastropod B. naticoidea is often the numerically dominant heterotroph in these mussel bed communities, along a wide geographic range (Carney 1994; Bergquist et al. 2005). This nerite grazes on the surface of B. childressi shells, ingesting free-living bacteria that are abundant on the shell surface, as well as on fragments of mussel periostracum and attached byssal fibers (Zande & Carney 2001). The size structure of B. naticoidea populations is often similar to that of the mussels in the mussel bed where they occur (Zande & Carney 2001), suggesting a connection between factors influencing the snail and mussel populations. Moreover, fossil records of a Miocene-age cold seep show a neritic species similar to B. naticoidea found with a Bathymodiolus-like methanotrophic mussel (Taviani 1994), further demonstrating a strong association between these taxa. In light of B. naticoidea’s strong fidelity toward B. childressi, we predicted that the nerite might recognize chemical cues associated with this mussel species, enabling it to locate its preferred habitat. Therefore, we analyzed the behavior of live nerites exposed to potential chemical and biogenic cues using flow-through Y-maze experiments. As B. childressi beds are associated with elevated methane levels (Nix et al. 1995; Smith et al. 2000), we tested methane as a potential cue. Moreover, we analyzed the behavior of snails exposed to conspecific cues, as well as metabolites originating from B. childressi.

Material and Methods

  1. Top of page
  2. Abstract
  3. Problem
  4. Material and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References

We collected individuals of B. naticoidea and B. childressi using the Johnson Sea-Link submersible from Brine Pool NR-1 (27°43′24′′ N, 91°16′30′′ W; ∼650 m depth), a hydrocarbon seep site located on the upper Louisiana slope of the Gulf of Mexico. We transported them alive to the surface in a temperature-insulated box and maintained them on board the ship in buckets of chilled, well-aerated seawater. We changed the seawater in the buckets daily and bubbled it twice daily with methane to ‘feed’ the mussels. We brought the mussels and snails back alive to the laboratory at Pennsylvania State University and maintained them together in an aquarium at 6 °C and under atmospheric pressure. For routine aquarium maintenance as well as for experimental treatments, we used synthetic seawater (SSW) made using Reef Crystals (Aquarium Systems Inc.). We bubbled the SSW in the maintenance aquaria with methane four times a day, and filtered and aerated it using a flow-through filtration system. We replaced approximately 10% of SSW in the aquaria with freshly made SSW once a week. For control experiments with Mytilus edulis, we used mussels harvested from Orwell Cove, PE, Canada, and purchased from a local seafood market.

We observed snail behavior in a cold room (6 °C) using a flow-through Y-maze system. The mazes consisted of glass Y-shaped tubes (with three 9-cm long arms and 1.6 cm inner diameter), through which a flow of SSW was maintained at a rate of about 15 ml·min−1 (or 0.125 cm·s−1) using a peristaltic pump. Two arms of the Y-maze received flow from separate 5-gallon reservoirs, one of which contained a test cue, and the other a control cue. We used dye tests to confirm that there was no mixing between solutions in the arms containing the test and control cues, and the flow rate in these arms was equal. For each trial, we introduced one snail into the third arm (‘entry arm’) of the Y-maze. To remove directional bias, we introduced the test cue alternately on the left or right side of the entry arm. We washed the Y-mazes and tubing thoroughly between experiments to remove any residue of the introduced chemical cues and snail mucous. Table 1 describes how we prepared the cues for Y-maze treatments and defines the abbreviations we used in the remaining text.

Table 1.   A description of the experimental treatments, with abbreviations used to refer to them in the text and figures. All incubations were performed at 6 °C with 10 gallons of continuously aerated synthetic seawater (SSW) kept in a glass tank. N = number of trials (one snail per trial). In all treatments except for the methane treatment, the experimental cues were made in air-saturated seawater containing ∼325 μM oxygen. For the MES treatment, we incubated SSW with 10 M. edulis individuals, as they were similar in mass (within 1 g) to five large B. childressi individuals (incubated with SSW to make the BCS cue). Moreover, for the BNS treatment, we incubated SSW with 25 snails, as we counted an average of 25 snails on the shells of five large B. childressi mussels in the holding aquarium. For filtration treatments, i.e. 0.4_BCS and 0.2_BCS, fresh batches of BCS (distinct from the batches used for the BCS treatments) were made before filtration was performed.
Treatment (abbreviation)NDescription of cues
TestControl
Methane23SSW with ∼300 μM dissolved methane and ∼150 μM dissolved oxygenSSW with ∼150 μM dissolved oxygen (achieved by bubbling nitrogen gas)
B. childressi seawater (BCS)47SSW incubated with 5 large mussels (>60 mm shell length) for 5 days, bubbled with methane once a daySSW alone incubated for 5 days and bubbled with methane once a day (CS)
M. edulis seawater (MES)10SSW incubated with 10 M. edulis mussels for 5 daysSSW alone incubated for 5 days
B. naticoidea seawater (BNS)19SSW incubated with 25 snails for 5 daysSSW alone incubated for 5 days
15d_BCS18Same as BCS, except mussels were maintained for 15 days with water changes on days 5 and 10. The SSW incubated with mussels for the last 5 days was used as the test cueCS
0.4_BCS10BCS passed through a 0.4 μm pore filterCS passed through a 0.4 μm pore filter
0.2_BCS8BCS passed through a 0.2 μm pore filterCS passed through a 0.2 μm pore filter
AC_BCS17BCS autoclave treated at 15 pounds of pressure, 120 °C temperature, cooled, and filtered through cheeseclothAutoclave treated CS, cooled and filtered through cheesecloth
S_BCS11Same as 15d_BCS, except mussel shells were scrubbed with a wire brush before water changesCS

We prepared the cues fresh for each batch of experiments, which consisted of five trials each. There was a lot of inter-individual variability in the time it took the snails to traverse the arms of the Y-maze. Some experimental individuals moved immediately after they were introduced into the Y-maze, whereas others took up to 50 min to traverse the entry arm of the maze. As snail movement was slow, we observed their behavior for a total of 100 min and noted the position of each snail 10 times (once every 10 min), taking the first reading after the snail had traversed three-fourths of the entry arm for the first time. We removed trails from the analysis if the experimental animals had not moved from the entry arm for the entire duration of the experiment; this occurred in 22% of the total number of trials.

We analyzed nerite behavior using two different parameters. First, we determined the ‘initial arm choice’ from the percentage of snails that entered either the test or the control arm first, and used a binomial test to determine if the probability of choosing the test arm first was significantly greater than half (P < 0.05). Second, we determined the ‘proportion of time’ the snails spent in either the test or control arm. We defined the proportion of time spent as the total number of times, among 10 measurements, that a subject was in that arm. If the data were normally distributed (which was true for all treatments except for MES), we used a one-sided paired t-test to analyze differences between proportions of time spent in the test and control arms. If the data were not normal, we used a Wilcoxon signed rank test. We performed all statistical analyses using the program Minitab (Minitab Inc., State College, PA, USA).

Results and Discussion

  1. Top of page
  2. Abstract
  3. Problem
  4. Material and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References

Bathymodiolus childressi require methane as a substrate for their nutritional symbionts (Childress et al. 1986; Cary et al. 1988; Streams et al. 1997) and are typically found in large beds associated with elevated methane levels (Nix et al. 1995; Smith et al. 2000). Smith et al. (2000) found methane levels of 42–626 μmol·l−1 among the mussel beds at our study site. Therefore, B. naticoidea could potentially use methane as a cue to locate these beds. In this study, we used methane dissolved in seawater as a test cue in Y-maze experiments with nerites. We found that the nerites showed no preference for methane in these experiments (P = 0.661; binomial test; Fig. 1; P = 0.875; one-sided paired t-test; Fig. 2). On the other hand, nerites showed a strong preference for seawater acclimated with B. childressi. Sixty-two percent of the snails entered the arm containing Bathymodiolus-conditioned seawater (BCS) first (P = 0.039; binomial test; Fig. 1), and the time they spent in this arm was significantly greater than that in the control arm (P < 0.0001; one-sided paired t-test; Fig. 2). The attraction the nerites displayed toward B. childressi was apparently species-specific, as they showed no preference for seawater acclimated with the shallow-water mussel M. edulis (P = 0.623; binomial test; Fig. 1, and P = 0.541; one-sided paired t-test; Fig. 2).

image

Figure 1.  ‘Initial arm choice,’ or percentage of snails that entered either the test or the control arms of the Y-maze first. Bars with asterisks show significant (P < 0.05) preference for the test arm. Sample sizes used to calculate the initial arm choice are as mentioned in Table 1.

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image

Figure 2.  The mean ± SE of the proportions of time spent by snails in the test or control arms. Bars with asterisks indicate significant difference (P < 0.05) between times spent in the test and control arms. Experimental snails typically moved back to the entry arm of the Y-maze after they had explored the experimental and/or control arms. This led to relatively low proportions of time spent in the experimental and control arms.

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Individual nerite behavior is strongly influenced by the activities of other conspecifics that routinely crawl on B. childressi shells, depositing pedal mucous, feces, and egg cases. We established that they follow mucous trails of conspecifics, as snails chose the same arm of a Y-maze as a previous snail with 100% fidelity (six trials with mazes not washed between introduction of snails; data not shown). Thus, B. naticoidea’s attraction to BCS could be due to conspecific cues emanating from the mussel shell surface. We examined this possibility using two sets of experiments. First, we tested the behavior of snails exposed to seawater acclimated with only conspecifics, excluding mussels (BNS). We found that they showed no preference for BNS (P = 0.500; binomial test; Fig. 1, and P = 0.255; one-sided paired t-test; Fig. 2). Second, we tested seawater incubated with B. childressi kept separate from snails for 15 days, along with intermittent water changes (15d_BCS). This treatment should have reduced snail residue on the mussel shells. Initial choice for the 15d_BCS arm was not significant (P = 0.119, binomial test; Fig. 1). However, 61% of the snails entered the 15d_BCS arm first, which is the same percentage of snails that chose the BCS arm first. The lack of significance in the binomial test for the 15d_BCS treatment could be due to the relatively small sample size (18 trials), when compared with the BCS treatment (47 trials). Moreover, the snails spent significantly greater time in the 15d_BCS arm than in the control arm (P = 0.0014; one-sided paired t-test; Fig. 2). Taken together these results indicate that B. naticoidea is attracted to a component in BCS that originates from B. childressi, and not from conspecific snails.

We further conducted a series of experiments to deduce the nature of the attractant. First we investigated whether the cue was particulate. We filtered BCS through 0.2 or 0.4 μm pore-size filters before using it in Y-maze treatments (0.2_BCS and 0.4_BCS, respectively). These treatments likely eliminated most fragments of periostracum or byssal threads and bacteria originating from mussel shells, which are 0.5 to 20 μm in diameter (Zande & Carney 2001). The snails had a strong preference for both 0.2_BCS and 0.4_BCS (Figs 1 and 2), implying that the cue that attracts them to BCS is most likely not an intact particle. Instead, it is a solute or particle less than 0.2 μm in size. Further, we autoclaved the BCS to denature proteins and break down complex carbohydrates (AC_BCS). Only 53% of the snails entered the AC_BCS arm first (P = 0.315; binomial test; Fig. 1), but the snails still spent significantly more time in this arm than in the control arm (P = 0.046; one-sided paired t-test; Fig. 2). Thus, the attractant in BCS is heat sensitive but its effect is not abolished by autoclave treatment. This result is consistent with a complex cue, such as a combination of a heat-sensitive peptide or complex carbohydrate, and a heat-insensitive component such as an amino acid or sugar. Both simple sugars and amino acids have been implicated as food-locating cues for shallow-water snails (Lombardo et al. 1992; Krug & Manzi 1999). Alternately, the cue could be a relatively small peptide whose activity is only partially suppressed by heat treatment. Low molecular weight peptides are a widespread type of cue used by aquatic organisms (Rittschof & Bonaventura 1986; Rittschof 1990). Further studies are needed to identify the active component of BCS.

Finally, as the nerites graze on the organic film on B. childressi shells, we hypothesized that the attractant in BCS originates from this film. We tested this by scrubbing B. childressi shells with a wire brush, likely removing most of the organic film, before using seawater incubated with them as a cue (S_BCS). In accordance with our premise, snails showed no preference for S_BCS (P = 0.274; binomial test; Fig. 1, and P = 0.129; one-sided paired t-test; Fig. 2). Due to the relatively low power of our statistical analysis, we cannot say for certain whether scrubbing the mussel shells abolished snail attraction to mussel cues completely. However, our preliminary findings indicate the signal that attracts B. naticoidea in BCS likely originates from the organic film on B. childressi shells. Consistent with our findings, B. naticoidea showed a strong preference to aggregate on B. childressi shells when presented with periostracum-covered and recently emptied B. childressi and Mytilus shells (A. Van Gaest, personal communication).

Our study demonstrates that B. naticoidea can perceive and respond to cues associated with B. childressi, and the cue likely originates from organic material covering shells of live mussels. The seafloor at our study site located at 650 m depth in the Gulf of Mexico typically experiences currents with average velocities of 10 cm·s−1 (Welsh & Inoue 2000). The flow rates we used in our study (0.125 cm·s−1) were substantially lower than these currents. Thus, it is feasible that seafloor currents can transport cues that the nerites can use to locate active B. childressi mussel beds. This could be important if the snails wander off from mussel beds, or if mussels in the bed they occupy die. Changing seepage patterns can lead to substantial temporal and spatial variability in the growth and physiological condition of mussels occupying seep sites in the Gulf of Mexico (Nix et al. 1995). Significant differences in physiological condition of mussels have also been observed within different areas of a single mussel bed, such as the one surrounding the Brine Pool NR-1 (Smith et al. 2000). Thus, B. naticoidea’s ability to locate live mussels, and thereby its food source, could be important for its survival in its natural habitat. This mechanism for detecting mussel beds may also be used for settlement by B. naticoidea larvae, but further studies are needed to examine this possibility. Chemicals associated with chemosynthetic environments are commonly thought to be attractants for organisms endemic to them. For example, sulfide was implicated as a possible attractant for vent endemic shrimp (Renninger et al. 1995). In our study, we found that a cue originating from habitat providing mussels, and not methane, evoked an attraction response from B. naticoidea.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Problem
  4. Material and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References

We thank the captain and crew of the Research Vessel (RV) Seward Johnson II as well as the crew and pilots of the Johnson Sea Link submersible (Harbor Branch Oceanographic Institution). We would also like to thank the LSU College of Basic Sciences Glass Shop for making the Y-mazes. The Minerals Management Service, Gulf of Mexico Regional OCS office, through contract number 1435-01-96-CT30813, the NOAA National Undersea Research Program at the University of North Carolina, Wilmington, and the National Science Foundation grant OCE 0117050 supported this work.

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  1. Top of page
  2. Abstract
  3. Problem
  4. Material and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References
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