Olfaction and antennule flicking rates in the American lobster (Homarus americanus) in response to contact with an odor plume

Alex Adams

Professor Bob Morris

Advanced Marine Biology 331

Wheaton College

Web posted on May 3, 2005

Abstract

This study focused on olfaction in the American lobster, Homarus americanus, the primary sense used by the benthic decapod for orientation and survival in the marine environment. This study examined the rate of antennule flicking when the lobster was exposed to a series of odor plumes. Three odorants and one control were used in this study and were dyed red to make them visible in the water. Video analysis showed antennule flicking rates over the course of the trial as well any change in flicking rate before and after the odor plume made contact with the antennules. Results showed that there was no significant difference between flicking rates before and after contact with antennules in the control trials, Mytilus edulis odorant trials, or brine shrimp odorant trials. A significantly higher flicking rate was found after contact in trials using the odorant of another lobster. Results also show that when smelling another lobster, antennules show an initial response and secondary response. This research suggests antennule flicking is related to odor detection.

Introduction

The ability of the American lobster (Homarus americanus) to detect an odor plume in the marine environment is crucial to the organism's survival (Horner et al., 2004). Olfaction in H. americanus is the primary sense used to locate mates and food items (Horner et al., 2004). Similar to many other benthic, decapod crustaceans, lobster olfaction is also necessary for predator avoidance, competitor recognition and settlement selection (Boudreau et al., 1993,). The ability to detect and recognize an odor plume in the water column is largely a product of the lobster's chemosensory anatomy (Romanowsky, 2000).

The anatomy of a lobster's olfactory organs consists of two sets of antennae (Romanowsky, 2000). The larger of the two antennae are used for olfaction and spatial orientation (Leonard et al., 1994). The second smaller set of antennae is the primary means by which a lobster senses odorants in its environment. These antennas are called antennules (Romanowsky, 2000). The antennules are "Y" shaped olfactory organs that consist of three segments that bear tufts of small hairs that resemble the bristles on a tooth brush (Heuss, 2002, Romanowsky, 2000). The tufts of hair, called guard hairs, are arranged in a zigzag pattern along the antennules and are covered with chemoreceptive nerve cells (Heuss, 2002).

To understand olfaction in the American lobster, it is necessary to understand how the olfactory antennules function hydrodynamically. The speed at which antennules are flicked through the water greatly affects their Reynolds number (Moore and Crimaldi, 2004). Previous research has shown that antennule flicks in H. americanus have a faster down stroke than upstroke (Goldman and Koehl, 2001, Moore and Crimaldi, 2004). This research has also shown that a fast down stroke of the antennules causes the water to flow between the guard hairs on the antennules as opposed to being displaced around the guard hairs (Goldman and Koehl, 2001, Koehl et al., 2001). This "leaky down stroke" as termed by Mimi Koehl, UC Berkley, allows for odor molecules in the water to pass through the chemoreceptive guard hairs on the antennules where the odor is picked up and transmitted to the brain (Heuss, 2002). Unlike the fast down-stroke of the antennule's flick, the slow up-stroke is not moving fast enough to allow the water to pass between the guard hairs. Therefore the antennules act as paddles displacing water and the old odorants with new ones on the slow up-stroke (Heuss, 2002).

A second factor that is considered by this study is the properties of an odor plume in salt water. An odor plume is a dynamic, three-dimensional cloud of varying odor concentrations (Moore and Crimaldi, 2004). The concentration of odorant in a plume is largely dependant upon the rate at which the molecules diffuse through the water (Moore and Crimaldi, 2004). Research has shown that most molecules in water have a diffusion rate on the order of 10^-9m²/s^-1 (Moore and Cimaldi, 2004). Therefore odorant diffusion is a slow process over centimeter distances in zero turbulence (Moore and Cimaldi, 2004). The rate at which odor molecules diffuse in saltwater was considered in this study when determining the length of trials.

The behavior observed and quantified in this study is the flicking of antennules through the water in response to an odor plume. When a lobster smells the water for food, a mate, or a predator, the antennules flick up and down, sampling the odor in the water (Koehl et al., 2001). For this study, one flick is defined as a movement of the antennules vertically in relation to the lobster's body of any distance greater than 1 millimeter. One flick includes the downward and upward motion. It is hypothesized that the antennule's flicking rate per second will differ before and after the antennules make contact with a dyed odor plume.

This project is a collaborative effort between the students of Advanced Marine Biology at Wheaton College. Data gathered in this study and gathered by other studies conducted by the Advanced Marine Biology class in the months of March and April, 2005, illustrates comparisons in adaptations to the marine environment between different marine organisms. A study conducted by Allison Roca titled, "Prey Sensory in Sea Star Asterias Forbesi," provided comparisons in methods of odor detection between lobsters and sea stars. Results of Roca's study provide further comparisons between the evolutionary adaptations for stimulus recognition in lobsters and sea stars as a result of the stresses in their two different habitats (Roca, 2005). A report written by Maris Madeira titled, "Phototaxis vs. Coverage Preferences in Hemigrapsus sanguineus" shows similarities between decapod crustacean's responses to stimulus. (Madeira, 2005). Similar to the results of this study, Madeira found that decapod crustaceans are capable of showing a behavioral a preference to stimuli.

Methods and Materials

Animals

For this experiment, one male and one female American lobster were purchased from a local supermarket in Norton, Massachusetts one day prior to experimentation. Both lobsters were weighed, using a butcher's scale, at 1.00 pounds. Lobsters were transported to a laboratory at Wheaton College in Norton, Massachusetts where they were placed into a 20 gallon salt water tank located in a room maintained at 12 degrees Celsius. The temperature of the tank remained constant at 12 degrees Celsius for the duration of experimentation. The salinity of the tanks was also maintained at 35 parts per thousand using artificial salt water (Instant Ocean) (Horner et al., 2004). Lobsters were given for 24 hours adjustment time in the holding tank before any experiments were conducted to allow for acclimation. In this experiment, only the male lobster was used in trials for antennule observation. The female lobster was used only for the production of odorant solutions.

Odorant Creation

This study utilized three separate odorants and one control. The three odorants created for this experiment were brine shrimp (Artemia salina) concentrate, a concentrate made from the Common mussel, Mytilus edulis, and an odorant made from lobster (Homarus americanus) concentrate. Each of these odorants was created using the nearly identical methods with exception to the odorant made to represent the odor of another lobster.

To make the odor of M. edulis, 10 grams of dead mussel was chopped finely in 100ml of de-ionized water. The solution was then shaken until thoroughly mixed. The solution was then poured through cheese cloth to remove the mussel tissue from the solution (Horner et al., 2004). Forty-five drops of red food coloring was then added to the solution and the beaker was shaken thoroughly (Goldman and Patek, 2002). The odorant was then tested for neutral buoyancy using a graduated cylinder filled with sea water. If the odorant was too buoyant, sea salt (Instant Ocean) was added until buoyancy was naturalized. Fresh water was added to neutralize the buoyancy of negatively buoyant odorant solutions.

To make the odor of Brine Shrimp, 10 grams of concentrated brine shrimp were added to 100ml of de-ionized water in a beaker. The solution was shaken and then poured through cheese cloth to strain out the brine shrimp tissue (Horner et al., 2004). Forty-five drops of red food coloring was then added to the brine shrimp solution. The odorant was tested for neutral buoyancy and salt or fresh water was added as necessary to neutralize the buoyancy of the odor plume in the water.

The procedure for extracting lobster odorant required 100ml of de-ionized water. The female lobster was removed from the holding tank and placed into a large glass dish. Using a 2ml transfer pipette, de-ionized water was run over the lobster's mouth parts and reproductive organs and captured in the large glass dish (Cowan, 1991, Horner et al., 2004). Water was pipetted until all 100ml had been used. This procedure was repeated three times. Forty five drops of red food coloring was then added and the solution was shaken thoroughly. The solution was then adjusted to neutral buoyancy.

A control solution was also created using sea water with a salinity of 35 parts per thousand. Forty-five drops of food coloring were added and the solution was mixed well. Both the odorants and the control were kept in a refrigerator when not in use.

Experimentation

All experimental trials were conducted in a 20 gallon tank with a water tight barrier that divided the tank equally into two halves. Before experimentation, water temperature and salinity in the experimental tank was made match the holding tank to prevent shock to the animal that could interfere with the experiment. To prevent turbidity in the water, the experimental tank did not use a charcoal filter. An aerator was placed into the tank between trials to assure that the water remained oxygenated, and it was removed five minutes prior to experimentation to allow the water to calm. All sides with exception to the front side of the tank were covered in white paper to eliminate peripheral visual distractions for the test animals. All experimental trials were recorded using a Panasonic mini DV digital video camera. Trials were recorded at 30 frames per second and lasted two minutes. Before the lobster was introduced to the experimental tank, the camera was set up with the lens 14cm from the glass of the tank and the zoom set to the widest angle possible. The male lobster was then introduced into the experimental tank and allowed at least five minutes to acclimate before experimentation began. Before recording, the camera was finely adjusted to assure that antennules were in clear view on the video camera screen and body movement had stopped.

An experimental trial began when camera began recording. Each trial was run for 15 seconds before adding an odorant. In each trial, the method for adding the odorant to the water was the same. 2ml of odorant was pipetted into the tank using a plastic transfer pipette. The odorant was injected into the water no more than 15 cm away from the lobster and the plume was allowed to disperse throughout the tank. Each trial lasted for 2 minutes, which was enough time for the dyed odorant to completely disperse throughout the water in the experimental tank. Between trials, odorized water was changed with clean seawater. With each water change, temperature and salinity were adjusted to match that of the holding tank. For each odorant, three trials were conducted.

Quantification

In this experiment, the lobster's response to a dyed odorant was measured in antennule flicks per second. Using the recorded digital film and the program iMovie for Macintosh computers running in the Imaging Center for Undergraduate Collaboration (ICUC) at Wheaton College, antennule flicking rates were determined at two points during the two minute experimental trial: in the first fifteen seconds and the last fifteen seconds of each trial. The flicking rate was determined by counting the number of flicks in a fifteen second time period and then dividing that number by 15 seconds (Number of flicks/15 seconds = flicking rate/second). Fifteen seconds was arbitrarily chosen as the sample period because it was long enough to determine an accurate rate of flicks per second and short enough to show any changes between initial and final rate. One flick was defined as a movement of the antennules vertically in relation to the lobster's body of any distance greater than 1 millimeter. One flick includes the downward and upward motion. Flicking data was gathered by observing the flick of only one of the two antennules. This experiment focused only on the Lobster's left antennule for all trials since the right antennule was damaged. Data of flicks per second was averaged over all trials with each odorant and graphed using a bar graph that showed that comparisons between the rate at the beginning of the trial, before exposure to an odorant, and at the end, constant exposure to the odorant (Figure 1). A student's t test assuming unequal variances was conducted on each group of trials for each odorant to test for a significant difference in antennule flicking rate before and after contact with an odor plume (Table 1).

Further data analysis was conducted to assess if there was a change in antennule flicking rate at the point at which the dyed odor plume makes contact with the antennule. Antennule flicks per second were calculated every ten seconds over the duration of a trial. This analysis was conducted only on three trials in which the odorant of another lobster was used.

Results

The results of the analysis of a lobster's antennule flicking rate in response to contact with an odorant of the common mussel and of brine shrimp indicate flicking rates do not vary significantly before and after contact (Table 1, Figure 1). This experiment showed that a lobster flicks its antennules significantly faster when the odorant of another lobster is in contact with its antennules compared to the flicking rate when no odorant is present (Table 1).Control trials showed no significant difference in flicking rates before or after antennule contact with an odorant (Table 1, Figure 1). Trials conducted using the Common mussel odorant and the brine shrimp odorant show similar results to the control indicating no significance was found between initial and final flicking rates (Table 1, Figure 1).

Further analysis of the three significant trials using the odorant of another lobster show flicking rate increased in an initial response and then in a secondary response (Figure 2, 3). In two of the three trials with the odorant of another lobster, the secondary response showed a higher flicking rate then the initial response (Figure 3). The average flicking rates before and after contact with brine shrimp odorant show a large variation in the rates before and after contact though the difference was not significant (Table 1, Figure 1).

Figure 1.  This figure shows a comparison between average flicking rates in the initial fifteen seconds and the final fifteen seconds of the four trials conducted. For each odorant a minimum of three trials were conducted. One trial lasted two minutes. There is significant difference only in trials with lobster odorant.

Table 1. This table shows the P values determined by a t test assuming unequal variances. The initial and final antennule flicking rates were averaged for each trial and compared for significant difference. An * denotes a significant difference (P value <.05) between initial and final flicking rates for an odorant.

Figure 2. This figure shows the average flicking rate over three trials in which the lobster was exposed to the odorant of another lobster. Flicking rate was recorded every ten seconds for the duration of the two minute trial.

Figure 3. This figure shows the variation between the three trials conducted using the odorant of another lobster. Values are recorded in percent maximum.

Discussion

Olfaction in the American lobster, Homarus americanus, plays a large role in orientation and survival in their environment (Horner et al., 2004). The purpose of this experiment was to see if antennule contact with an odor plume caused a change in flicking rate. This study proposes that a change in flicking rate may be an indicator of a lobster's recognition of an odorant in the water column. The working hypothesis in this study is that antennule flicking rate in the American lobster will differ before and after the antennules make contact with an odor plume. The results of this study supported the hypothesis but this dependant upon the type of odorant tested. In support of the hypothesis, results show a lobster exhibits a significant change in antennule flicking rates over three trials when its antennules come in contact with the odorant of another lobster. Contrary to the hypothesis, there was no significant change in antennule flicking rates when antennules came in contact with the odor of Mytilus edulis, or brine shrimp (Table 1, Figure 1-3). Analysis of the control shows that there is very little difference, with no significance, between the average flicking rates over the four trials run using only dye and no odorant. This data supports the null hypothesis, that there will be no difference between flicking rates before and after the odorant makes contact with the antennules. Based on the data very similar data before and after contact with the control, it can be hypothesized that in response to a plume consisting of only food coloring, there is a "normal" flicking rate with insignificant differences before and after contact (Table 1, Figure 1).

Numerous studies have investigated interactions between lobsters and the role of olfaction in these interactions. The results of this study may indicate further data that supports research showing intra-species, chemical interactions and reception (Cowan 1991). In previous studies on lobster olfaction, it has been shown that some species of lobsters have methods of smelling the water that are unique to their species (Goldman and Patek, 2002). Such differences involve the rate at which the antennules move though the water, the duration of a flick, and the distance covered by a flick (Goldman and Patek, 2002).

The results of this study, indicating an initial and secondary response, may represent the American lobster's method of smelling the water over time (Figure 2, 3). Further speculation on the biological basis behind an increase in flicking rate is that H. americanus may indicate an optimal odorant concentration at which the guard hairs on the antennules are best able to extract the odorant from the water. A second possibility is that the anatomy of guard hairs along the antennules may be more susceptible to specific odorant chemicals over others (Moore and Crimaldi, 2004). A second aspect that must be considered when speculating on the biological reasoning behind the significant change in flicking rates is the potential for chemical message reception. The lobster odorant used in this study was taken from a female lobster. There is substantial data that suggest that lobsters communicate via chemical odorants released in urine (Cowan, 1991). The significant results shown for the trials using the odorant of another lobster suggest that there is in fact chemical reception taking place as indicated by the change in rates of antennule flicking before and after contact with the lobster odor plume (Table 1, Figure 1, 2, 3) (Cowan, 1991, Roca, 2005). Based on the data and the experiments run, it is possible that the results shown are a product of the olfaction of the opposite sex, although these data may simply be a normal response to another lobster.

Trials conducted with common mussel and brine shrimp odorants indicate insignificant differences before and after odorant contact with antennules. By rejecting the hypothesis, this data may suggest several things. It is possible that the American lobster's method of olfaction varies with the odorant. The detection of brine shrimp odor and common mussel odor may not involve a change in flicking rate. Furthermore, despite evidence to suggest otherwise, it is possible that flicking rate is not an indication of odor reception of the odorants used in this study (with exception to that of another lobster) and the lobster may be using a completely different sense to detect odor in the water. Another possibility is that there was no change in flicking rate when contact with an odorant was made as a result of error.

The possibility of error was considered in this study. One source of error that was acknowledged by in this research is lobsters may not be able to detect the odorant as a result of the properties of the created odorant, the scientific methods or the quantity administered in the trials. Roca determined in 2005 that sea stars are able to detect both prey odors and non-prey odors indicating that some marine organisms have olfactory capabilities that extend beyond their normal function (Roca, 2005). A second source of error pertains to the binding of dye molecules to odor molecules. It is possible that odorants diffused at different rates than the dye thus altering the point of odor contact. This error would affect the flicking rate before and after contact with a dyed odor plume (Figure 1). The lobster used in this experiment had one damaged antennule. Although the damaged antennule was not focused on for quantification in this study, its deficiency may have caused the second, intact antennule to flick at an abnormal rate to compensate thus affecting the rate data. Another possibility is that the American lobster is using a different organ than the antennules to detect odor in the water. One possibility is the larger pair of antennas (Romanowsky, 2000). Finally, the experimental conditions presented the possibility of the lobster being disturbed during a trial run by movement outside the tank. It is unknown whether lobster's flicking rate varies in response to visual stimulus though research by Madeira in 2005 has shown decapod crustaceans exhibit a tactic response to changes in light. Therefore, shadows and movements outside the tank are a potential source for error in this study.

Further research on this topic should consider several modifications to the experimental method. The trial period in this experiment lasted two minutes. The results show that in all three trials with lobster odorant, the highest or second highest observed flicking rates occurred in the last ten seconds of the trial (Figure 2, 3). This suggests the rate may continue to increase, may decrease and it is possible that a third peak response may occur given more time. A follow up study should lengthen trial periods to observe flicking rates in decreased odor concentrations that extend beyond two minutes. Secondly, results with brine shrimp odorant show nearly significant data suggesting that the initial flicking rate is greater than the final flicking rate, contrary to the hypothesis. More trials run with brine shrimp may show significance (Table 1, Figure 1)

In conclusion, this study sheds light on the method of olfaction used by the American lobster Homarus americanus. The results of this study show the American lobster has a no significant change in antennule flicking rates before and after contact with the odor of brine shrimp, a non-prey item, and Mytilus edulus, a common prey item. However this study did show there was significant difference in flicking rates before and after a contact when exposed to the odor of another lobster. Furthermore, a profile of the flicking rate over time when the lobster is exposed to the odorant shows that flicking rates oscillate between high and low number of flicks per second: an initial and a secondary response. The data gathered by this study serves as a starting point for more extensive research on the topic of odor recognition. Further studies should look at the rates at which antennules flick in response to a wider variety of odors, the phases of odor detection. A good goal for further research is to create a behavioral profile showing the characteristics of odor detection and recognition in the American lobster. The data gathered by this study in collaboration with two students from Wheaton College's Advanced Marine Biology show different adaptations to sensory reception in the marine environment.

Reference

Boudreau, B., Bourget, E., Simard, Y. 1993. Behavioural responses of competent lobster
postlarvae to odor plumes. Marine Biology: 117:1, 63-69.

Cowan D. F. 1991. The role of olfaction in courtship behavior of the American lobster
Homarus americanus. Biol. Bull. 181:402-407.

Goldman, J.A., Koehl, M.A.R. 2001. Fluid dynamic design of lobster olfactory organs:
high speed kinematic analysis of antennule flicking by Panulirus argus. Chem.
Senses. 26:385-398.

Goldman, J.A., Patek, S.N. 2002. Two sniffing strategies in palinurid lobsters. Journal of
Experimental Biology. 205: 3891-3902.

Heuss, C. 2002. Sniff. Sniff. How does a lobster's nose know? Science Notes: U.C.S.C
Science Communication Program.
http://scicom.ucsc.edu/scinotes/0201/lo/lobster/

Horner, A.J., Weissburg, M.J., Derby, C.D. 2004. Dual antennule chemosensory
pathways can mediate orientation by Caribbean spiny lobsters in naturalistic flow
conditions. Journal of Experimental Biology. 207: 3785-3796

Koehl, M. A. R., Koseff J.R., Crimaldi, J.P., McCay M.G., Cooper, T., Wiley, M.B.,
Moore, P.A. 2001. Lobster Sniffing: Antennule Design and Hydrodynamic
Filtering of Information in an Odor Plume. Science: 294.

Leonard, A. E., Voigt, R., Atema, J. 1994. Lobster Orientation in Turbulent Odor
Plumes: Electrical Recording of Bilateral Olfactory Sampling (Antennular
"Flicking")Biol. Bull. 187: 273.

Madeira, M. 2005. Negative Phototaxis vs. Coverage in Hemigrapsus Sanguineus.
Wheaton College.
http://icuc.wheatonma.edu/%7Ebio331/2005/mmadeira/index.html

Moore, P., Crimaldi, J. 2002. Odor landscapes and animal behavior: tracking odor plumes
in different physical worlds. Journal of Marine Systems 49: 55-64.

Roca, A. 2005. Prey Sensory in Sea Star Asterias Forbesi. Wheaton College.
http://icuc.wheatonma.edu/%7Ebio331/2005/aroca/index.html

Romanowsky, K. 2000. Overview of Homarus americanus: The American Lobster.
Young Canada Works in Heritage Institutions.
http://www.parl.ns.ca/lobster/overview.htm