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Biol. Bull. 215: 24-33. (August 2008)
© 2008 Marine Biological Laboratory

Identification of Chemosensory Sensilla Mediating Antennular Flicking Behavior in Panulirus argus, the Caribbean Spiny Lobster

Peter C. Daniel1,*, Michael Fox2 and Sagar Mehta3

1 Department of Biology, Hofstra University, Hempstead, New York 11549
2 Plainview Old Bethpage John F. Kennedy High School, Plainview, New York 11803
3 The Wheatley School, Old Westbury, New York 11568

* To whom correspondence should be addressed. E-mail: peter.c.daniel{at}hofstra.edu


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Crustaceans sample odorants by a rapid series of flicks of the two flagella composing the distal segments of each of the paired antennules. The lateral flagella contain aesthetasc sensilla that house unimodal chemosensory neurons. Nine types of nonaesthetasc setae with putative chemosensory and mechanosensory functions are distributed on the lateral and medial flagella. Sensory neurons in aesthetascs and nonaesthetasc sensilla terminate in separate regions of the brain, the olfactory lobe, and the lateral antennular neuropil, resulting in two odorant-processing pathways. Distilled water ablation of flagella and excision of specific setae were used to identify chemosensory sensilla mediating antennular flick behavior in Panulirus argus. The flick rates of sham-ablated and ablated or excised lobsters toward squid extract were compared. Complete attenuation of flick response to squid extract occurred as a result of (1) distilled water ablation of lateral flagella, (2) excision of aesthetascs and asymmetric sensilla, and (3) excision of aesthetascs. Distilled water ablation of medial flagella resulted in a mean flick rate 52% of that observed for sham-ablated lobsters toward squid extract. Flicking was unaffected by excision of asymmetric, guard, or companion sensilla. We propose that odorant mediation of flicking behavior requires both the aesthetasc and nonaesthetasc pathways.

Abbreviations: AGB, antennular grooming behavior • ASW, artificial seawater • DW, distilled water • LAN, lateral antennular neuropil • L-glu, L-glutamate • OL, olfactory lobe • SE, squid extract


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Literature Cited
 
Crustaceans possess an impressive diversity of setae on the surface of their exoskeletons, with a particularly large number located on the antennules, the first pair of antennae. The antennules have been identified as chemosensory mediators of a number of important behaviors in crustaceans, including courtship (Gleeson, 1982), individual recognition (Karavanich and Atema, 1998; Atema et al., 1999; Giri and Dunham, 2000; Johnson and Atema, 2005), antennular flicking (Daniel and Derby, 1991), antennular grooming behavior (AGB) (Wroblewska et al., 2002; Schmidt and Derby, 2005), and food odor recognition and tracking (Derby and Atema, 1982; Reeder and Ache, 1980; Giri and Dunham, 1999; Steullet et al., 2001, 2002; Keller et al., 2003; Horner et al., 2004). For some behaviors, a single sensillum type appears to mediate the behavior (Gleeson, 1982; Johnson and Atema, 2005; Schmidt and Derby, 2005; Horner et al., 2008a, b). For other behaviors, a number of different sensilla are necessary (Steullet et al., 2001, 2002; Horner et al., 2004). In this study, we focus on the role of antennular sensilla in mediating antennular flick behavior, a behavior that functions to enhance stimulus access to chemoreceptors on the antennules.

Two flagella compose the most distal segments of each antennule. A flagellum consists of repeating segments called annuli (Laverack, 1964; Steullet et al., 2000). Although both flagella contain setae, the most distal third of annuli on the ventral surface of the lateral flagellum contains a region dense with several types of setae found specific to that region, the so-called "tuft" setae (Laverack, 1964; Gleeson et al., 1993; Cate and Derby, 2001). In Panulirus argus, the Caribbean spiny lobster, this region consists of four types of setae: aesthetascs, asymmetric sensilla, guard setae, and companion setae (Fig. 1A). The aesthetascs are by far the most numerous of the four types. Approximately 1300 aesthetascs are present on both flagella, with about 16–24 arranged in two rows per annulus (Laverack, 1964; Gleeson et al., 1993; Cate and Derby, 2001). Each aesthetasc is innervated by about 300 olfactory neurons (Laverack and Ardil, 1965; Spencer and Linberg, 1986; Grünert and Ache, 1988; Steullet et al., 2000; Cate and Derby, 2001). The aesthetascs are the only chemosensilla known to have unimodal function. There is one asymmetric sensillum on each tuft annulus (for a total of ~80 sensilla), positioned such that it is along the lateral boundary of the two rows of aesthetascs with the shaft curving inward between the two rows. Asymmetric sensilla, like all other sensilla types with the exception of aesthetascs, are most likely bimodal, containing an indeterminate number of mechanosensory and chemosensory neurons (Schmidt and Derby, 2005). Guard setae (~160) flank both the medial and lateral edges of the aesthetascs, with two per annulus (Laverack, 1964; Spencer and Linberg, 1986; Gleeson et al., 1993; Cate and Derby, 2001). One or two companion setae are located near each guard seta. Guard and companion setae contain some sensory innervation that remains virtually undefined (Laverack, 1964).


Figure 1
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  		Figure 1. Examples of lateral flagella before and after some of the excision techniques (Excision Experiments: Part 2). Light micrographs. (A) Intact lateral flagellum. Each annulus contains medial and lateral guard and companion setae (GS and CS) flanking two rows of aesthetascs (AE). One asymmetric sensillum (AS) can be observed between the lateral edge of aesthetascs and the guard and companion setae. (B) Lateral guard, companion, and asymmetric setae excised. Behavioral assays were performed after removal of lateral guard and companion setae (sham) and again after removal of asymmetric sensilla. (C) Medial guard setae, companion setae, and aesthetascs excised. In this example, several aesthetascs remain. Behavioral assays were performed after removal of medial guard and companion setae (sham) and again after removal of aesthetascs.

 
Distributed along the lengths of both medial and lateral flagella, with the exception of the tuft region, are the "nontuft" setae. The most numerous are the hooded setae (~1200), followed by short setuled (~400), medium simple (~300), short simple (~300), plumose (~100), and long simple (~30) (Cate and Derby, 2001). Judging from their ultrastructure, hooded, medium simple, and long simple setae all appear to have bimodal sensory function. In terms of numbers, there are actually more sensilla with chemosensory function in the nontuft regions than in the tuft regions of the antennules. However, the level of innervation of these sensilla by chemosensory neurons is much lower—from 5 to 10 per sensillum (Cate and Derby, 2001).

The output from antennular sensilla is processed through two independent pathways in the central nervous system (Schmidt and Ache, 1992, 1996a, b; Schmidt et al., 1992). The olfactory neurons that innervate aesthetascs follow the "aesthetasc pathway"; they terminate on the glomeruli composing the paired olfactory lobes of the brain (OL). The "nonaesthetasc pathway" is composed of chemosensory and mechanosensory neurons that innervate all other sensilla, whether in the tuft or nontuft regions of the flagella, that terminate in the stratified paired lateral antennular neuropils (LAN). Different regions of the terminal medullae are innervated by interneurons ascending from the OL and the LAN (Sullivan and Beltz, 2001). Thus, even higher-order processing of the two pathways remains relatively distinct, with the exception that some local interneurons in the deutocerebrum appear to innervate both the OL and LAN (Schmidt et al., 1992; Mellon and Alones, 1995; Mellon, 1996; Schmidt and Ache, 1996b). The LAN and its neighbor, the median antennular neuropil (MAN), provide motor output controlling the movement of the antennules, including those involved in such behaviors as flicking, AGB, withdrawal, and waving (Maynard, 1965; Schmidt and Ache, 1993; Roye, 1994).

For some behaviors, a single type of sensillum may provide the chemosensory input mediating a specific behavior. Asymmetric sensilla are the sole source of chemosensory input mediating AGB (Schmidt and Derby, 2005). Some social interactions may be mediated solely by aesthetascs. Pheromone-mediated elicitation of courtship behavior in the blue crab, Callinectes sapidus, does not occur in the absence of aesthetascs (Gleeson, 1982). Recognition of opponents in agonistic encounters between crayfish, Procambarus clarkia (Horner et al., 2008b), and between American lobsters, Homarus americanus, also requires aesthetascs, although the ablation technique used in the latter study did not discriminate between aesthetascs and asymmetric sensilla (Johnson and Atema, 2005). Recognition of social signals involved in sheltering behavior in P. argus also requires aesthetascs (Horner et al., 2008a). Other behaviors apparently can be elicited by a number of sensillum types, so far rather broadly defined. Orientation to food odors, search behavior, odor-associated learning, and odorant discrimination can be elicited by either aesthetascs alone or nonaesthetasc sensilla alone (Horner et al., 2004; Steullet et al., 2001, 2002). Thus some chemosensory behaviors may use only one processing pathway, either the aesthetasc pathway (e.g., courtship, individual recognition) or the nonaesthetasc pathway (e.g., AGB), while other behaviors appear to use either pathway in an apparently redundant fashion (e.g., search behavior).

Crustaceans sample the chemical stimuli in their environments by rapidly flicking the flagella of their antennules (Snow, 1973; Price and Ache, 1977; Schmitt and Ache, 1979). In antennular flicking, the lateral flagellum sweeps downward toward the medial flagellum, decreasing the angle between the two flagella. This is followed by a slower upstroke, returning the lateral flagellum to its original position. During the downstroke the boundary layer around the closely spaced aesthetascs is reduced, thereby facilitating stimulus access (Koehl et al., 2001; Koehl, 2006). During the upstroke and before the next downstroke, the increase in boundary layer around the aesthetascs traps sampled chemicals long enough for them to diffuse into the sensilla lymph of the aesthetascs. One motorneuron drives contraction of the single muscle depressing the lateral flagellum (Mellon, 1997). The motor pattern can be activated by hydrodynamic stimulation of the body and both pairs of antennae as well as by odorant stimulation of the antennules. In P. argus, flick rates in the absence of stimuli range from 0.4 to 1.5 Hz (Gleeson et al., 1993; Goldman and Koehl, 2001). Antennular flicking increases by about 1 Hz in response to single chemicals found in food stimuli and is activated by chemoreceptors found on the antennules (Daniel and Derby, 1991). In this study, we used ablation techniques to determine which specific subsets of antennule sensilla may provide chemosensory input mediating antennular flick in P. argus. We found that aesthetascs are necessary for elicitation of antennular flick; nonaesthetasc chemosensilla also contribute.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Literature Cited
 
Source and maintenance of Caribbean spiny lobsters
Caribbean spiny lobsters, Panulirus argus Latreille, 1840, were acquired from the Florida Keys Marine Laboratory in Long Key, Florida. Lobsters were maintained in individual 80-1 aquaria with one lobster per aquarium, at 25–27 °C with a light-dark cycle of 12 h:12 h, and a practical salinity level between 34 and 37. Red light (25-W ceramic-coated light bulbs) was provided during data collection periods. Aquaria were equipped with gravel bottom filter systems; lined with crushed coral; and filled with aerated, recirculating Instant Ocean. Spiny lobsters were fed shrimp, squid, or fish every other day.

Ablation procedures
Ablation techniques used in this study included distilled water (DW) ablation and excision. In DW ablation, antennules are exposed to DW for a 15-min period. The higher osmotic potential inside the chemosensory neurons exposed to the water via the porous cuticle destroys the dendritic portion of chemosensory receptors in contact with DW (Gleeson et al., 1996). Excision allows removal of specific sensilla types. It destroys the dendrites of sensory cells that extend into the setae, resulting in loss of function, and eventually leads to the death of olfactory receptor neurons (Harrison et al., 2001).

DW ablation experiments.
These two experiments determined the relative roles of chemoreceptors on the lateral, medial, and both flagella in mediating flicking behavior (Table 1). After each ablation step, lobsters were allowed to recover for 1 h before being tested with chemical stimuli. Further ablation steps occurred no earlier than 24 h after the previous ablation. For all ablations, each lobster was removed from its tank, wrapped in wet paper towels, and restrained in an Instant Ocean bath. In the first experiment, a sham ablation (control ablation) was performed on 12 lobsters by placing both the medial and lateral in a 15-ml vial containing artificial seawater (ASW) (Cavanaugh, 1964) for 15 min. In the next ablation step the medial flagella were then placed in a 15-ml vial containing DW for 15 min. In the last step, both the medial and lateral flagella were immersed in the vial containing DW. In the second experiment a second sham ablation was performed on nine lobsters. The final step consisted of DW ablation of only the lateral flagella.


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  		Table 1 Protocols for ablation techniques and behavioral assays

 
Excision experiments.
Lobsters were restrained with ventral side up in a shallow container of Instant Ocean. The lateral flagellum of one antennule was immobilized by stapling to Sylgard in a small plastic container filled with Instant Ocean. Sensilla were excised under a dissecting microscope by cutting them off at their base, using a handcrafted tool. The tool was fashioned from a thin carbon-steel blade custom-shaped into a micro-scalpel by using a blade holder and breaker, and was attached by glue to an applicator stick. After each ablation step, lobsters were allowed to recover for at least 24 h before being tested with chemical stimuli. Further ablation steps occurred at least 24 h after the previous ablation step. In the two experiments conducted, sham ablations were performed in which the lobster was held in the restraining device for 45 min.

Excision experiments: Part 1. These two experiments focused on examining the role of tuft setae in flicking behavior by excising either all tuft setae or only aesthetasc and asymmetric sensilla (Table 1). In the first experiment, using nine sham-ablated lobsters, both lateral and medial rows of guard setae and companion setae were removed from the tuft region of the lateral flagella of both antennules. Later, all tuft setae were removed from this group of lobsters. In the second experiment, also using nine sham-ablated lobsters, aesthetascs and asymmetric sensilla were removed from the tuft region of the lateral flagella of both antennules, leaving only guard and companion setae.

Excision experiments: Part 2. These two experiments focused on the specific roles of aesthetascs and asymmetric sensilla in flicking behavior and antennular grooming behavior (AGB) (Table 1, Fig. 1). In both experiments, a sham ablation was performed in which the row of guard setae directly adjacent to either the asymmetric sensilla (lateral boundary of tuft setae) or the aesthetascs (medial boundary of the tuft setae) were removed, allowing access to these sensilla in the subsequent ablation step. In the first experiment, this was followed by ablation of the asymmetric sensilla. In the second experiment, the aesthetascs were removed.

The completeness of the excision procedures was evaluated after each ablation step by examining sensilla remaining on lateral flagella after each ablation procedure following behavioral assays. Lobsters were restrained as described before, and sensilla were counted using the dissecting microscope.

Behavioral assays
Preparation of chemical stimuli.
ASW was used as a control stimulus and to dilute odorant solutions. Squid extract (SE) was used as a stimulant for antennular flicking (Daniel and Derby, 1991), and L-glutamate (L-Glu) was used as a stimulant for AGB (Barbato and Daniel, 1997). To prepare the SE, 43.1 g of squid was blended for 3 min with 1000 ml of ASW. The solution was centrifuged (Model-TJ6, Beckman Coulter) at 550 x g for 5 min. The supernatant was vacuum-filtered twice using glass fiber filters (C6) and then once through a cellulose nitrate membrane filter (0.45 µm). This resulted in a 10 mmol l–1 solution of SE, assuming the same composition of squid extract as described in Carr et al. (1996). The ultrafiltrate was stored in 5-ml plastic solution tubes at –80°C until needed. Stock solutions of L-Glu (10 mmol l–1, pH 7.9) were prepared in ASW. The SE ultrafiltrate and L-Glu stock solutions were stored in aliquot samples at –80 °C until needed. On the day of an experiment, odorant solutions were thawed and diluted with ASW to 1 mmol l–1 (SE) or 0.5 mmol l–1 (L-Glu).

Presentation of stimuli.
Lobsters were equipped with a removable headset to allow for automatic presentation of chemicals at a constant distance from the antennules (Daniel and Derby, 1991). Headsets were attached to each lobster 1 h before presentation of stimuli to allow acclimation. A multiheaded peristaltic pump circulated tank water continuously (20 ml x min–1), via plastic flexible tubing, from the tank through the stimulus introduction tubing, allowing constant presentation of mechanical stimuli in the absence or presence of chemical solutions. Five-milliliter test solutions of ASW, SE, or L-Glu (for Excision Experiments: Part 2 only) were injected by 5-ml plastic syringes into the tubing through two-way stopcock valves. Stimuli were presented in triplicate in randomized order. Responses were video-recorded beginning 30 s before and ending 2 min after stimulus presentation using a digital CCD camera sensitive to low-level illumination (LTC 035/60, Phillips) connected to a time-lapse video recorder with real-time display (SVF 124, Sony). The camera was equipped with a TV zoom lens (5–60 mm, 1:1.6, Raymax) to better visualize antennular movements.

Data analysis and statistics
Tapes were replayed at 1/6 speed to allow counting of individual flicks. Pre-stimulus flick rates (Hz) were subtracted from post-stimulus flick rates to correct for baseline activity. For Excision Experiments: Part 2, AGB was measured as well, to verify the effectiveness of the ablation procedure. The numbers of antennule wipes occurring over a 2-min period after stimulus presentation were recorded. The flick rates or wipes over 2 min for the three presentations of ASW and SE were averaged for each lobster, and the mean response to ASW was subtracted from the mean response to SE. Therefore it is possible to have negative mean flick rates, as is apparent in some of the results (see Figs. 3 and 4a). However, negative flick rates were well within one standard deviation unit of zero (i.e., no change in flick rate). In ablation experiments in which three ablation types were compared, the mean responses were analyzed using one-way repeated-measures (RM) ANOVA. Post hoc multiple comparisons were performed using the Holm-Sidak method. For other comparisons, paired Student's t-tests were performed. All statistical tests were performed with statistics software (Sigmastat 3.0, Systat Software).


Figure 3
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  		Figure 3. Excision Experiments: Part 1. Flick rates (Hz) to squid extract are mean values ± standard deviation. (A) Bilateral excision of guard and companion setae (GS and CS) followed by excision of remaining tuft setae (n = 9). Results of one-way repeated measures ANOVA are shown. Letters represent outcome of Holm-Sidak post hoc pairwise comparisons. Different letters indicate that means were significantly different (P < 0.05). Excision of guard and companion setae had no effect on flick rates compared to sham excision (animal confined in excision apparatus). Removal of the remaining tuft setae (aesthetascs and asymmetric sensilla) resulted in a significant reduction in flick rates. (B) Bilateral excision of aesthetascs and asymmetric sensilla (n = 9). Results of the paired t-test are shown. Excision resulted in a significant reduction in flick rates compared to sham excision.

 

Figure 4
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  		Figure 4. Excision Experiments: Part 2. Bilateral excision of either aesthetascs (AE) (n = 5) or asymmetric sensilla (AS) (n = 8). Results of the paired t-test are shown above each pair of bars. (A) Flick rates (mean values ± standard deviation) to squid extract after excision. First pair of bars represents responses to sham ablation (lateral guard and companion setae excised) followed by excision of asymmetric sensilla. Excision of asymmetric sensilla resulted in no change in flick rates. Second pair of bars represents responses to sham ablation (medial guard and companion setae excised) followed by excision of aesthetascs. Excision of aesthetascs resulted in a significant reduction in flick rates. (B) Number of antennular wipes in 2 min after application of L-Glu (mean values ± standard deviation) after excision. First pair of bars represents responses to sham ablation (lateral guard and companion setae excised) followed by excision of asymmetric sensilla. Excision of asymmetric sensilla resulted in no change in number of wipes. Second pair of bars represents responses to sham ablation (medial guard and companion setae excised) followed by excision of aesthetascs. Excision of aesthetascs resulted in no change in number of wipes.

 

   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Literature Cited
 
After all sham ablation techniques for all three sets of experiments, the behavioral responses to stimuli was significantly greater than responses to artificial seawater (ASW) (paired t-tests, P < 0.05). The flick rate pooled for all experiments for sham-ablated lobsters was 0.01 ± 0.16 (mean ± standard deviation) Hz toward ASW and 0.54 ± 0.32 Hz toward squid extract (SE). The wipe rate for sham-ablated lobsters, pooled for the second excision experiment, was 1.0 ± 1.9 toward ASW and 15 ± 10 toward L-Glu.

Distilled water ablation experiments
Bilateral DW ablation of the medial flagella resulted in a decrease in mean flick rate to 52% of the mean flick rate after sham ablation (both antennules soaked in ASW) (Fig. 2A, RM ANOVA, Holm-Sidak test, P = 0.001). Further ablation of the lateral flagella (= both medial and lateral flagella ablated) reduced the mean flick rate to 10% of the post-sham ablation level (RM ANOVA, Holm-Sidak test, P < 0.001). The mean flick rate after ablation of both flagella was also significantly lower than the mean flick rate after ablation of the medial flagella (RM ANOVA, Holm-Sidak test, P = 0.004).


Figure 2
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  		Figure 2. Distilled water (DW) ablation experiments. Flick rates (Hz) to squid extract are mean values ± standard deviation. (A) Bilateral DW ablation of medial flagella followed by ablation of both flagella (n = 12). Results of one-way repeated measures ANOVA are shown. Letters represent outcome of Holm-Sidak post hoc pairwise comparisons. Different letters indicate that means were significantly different (P < 0.05). Ablation of the medial flagella resulted in a significant decrease in mean flick rate compared to the mean flick rate after sham ablation (soak in artificial seawater). Further ablation of the lateral flagella resulted in a further significant decrease in mean flick rate. (B) Bilateral DW ablation of lateral flagella (n = 9). Results of the paired t-test are shown. The mean flick rate after DW ablation of the lateral flagella was significantly decreased relative to the mean flick rate after sham ablation.

 
Bilateral ablation of only the lateral flagella resulted in complete loss of responsiveness to the stimulus (Fig. 2B, paired t-test, P = 0.002). Mean flick rate after ablation of the lateral flagella was 1.1% of the mean flick rate after sham ablation.

Excision experiments: Part 1
Removal of the guard and companion setae on the lateral and medial boundaries of the tuft region of lateral flagella resulted in no decrease in mean flick rate compared to the mean flick rate after sham ablation (lobster immobilized in retaining device) (Fig. 3A, RM ANOVA, Holm-Sidak test, P = 0.81). Additional removal of all the remaining tuft setae (aesthetascs and asymmetric setae) completely attenuated the mean flick rate (RM ANOVA, Holm-Sidak tests, all tuft setae removed vs. sham: P < 0.001, all tuft setae removed vs. guard and companion setae removed: P < 0.001). The removal of only aesthetascs and asymmetric setae after sham ablation also resulted in complete attenuation of mean flick rate (Fig. 3B, paired t-test, P < 0.001).

Ablation of aesthetascs resulted in loss of 99.5% ± 0.424% (mean ± standard deviation) of aesthetascs (total number of aesthetascs estimated as 1300 according to Laverack, 1964; Gleeson et al., 1993; Cate and Derby, 2001) and no loss of guard and companion setae. Ablation of guard and companion setae resulted in their complete removal and loss of 2.5% ± 0.31% of aesthetascs.

Excision experiments: Part 2
In these experiments, the flick and wipe responses after bilateral excision of either aesthetascs or asymmetric setae were compared to the responses after sham ablation (excision of guard and companion setae along either the medial (aesthetascs excision followed) or lateral (asymmetric setae excision followed) boundaries of the tuft region of the lateral flagella). Excision of the aesthetascs resulted in complete attenuation of flick rates (Fig. 4A, paired t-test, P = 0.008), while excision of the asymmetric sensilla resulted in no change (paired t-test, P = 0.63). In contrast, wipe rates were unaffected by excision of aesthetascs (Fig. 4B, paired t-test, P = 0.58), but they were reduced to 9.0% (paired t-test, P = 0.003) after asymmetric sensilla excision.

As a result of excision of the lateral row of guard setae, 10.9% ± 5.85% (mean ± standard deviation) of the asymmetric sensilla (total asymmetric sensilla determined from total number annuli) were removed accidently. Deliberate removal of the asymmetric sensilla resulted in the loss of 98.6% ± 1.03% of asymmetric sensilla and 13% ± 1.6% of aesthetascs (see Fig. 1B, for example). For the aesthetasc-excision protocol, removal of the medial row of guard and companion setae resulted in no loss of asymmetric sensilla, and further removal of the aesthetascs resulted in 25.3% ± 6.35% loss of asymmetric sensilla and 97.3% ± 0.577 % loss of aesthetascs (see Fig. 1C, for example).


   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Literature Cited
 
The results of these experiments on Panulirus argus definitively identify the aesthetascs as chemosensilla necessary for modulating antennular flicking. Complete attenuation of flick response to the chemical stimulus used in the flick assay occurred as a result of (1) distilled water ablation of the lateral flagella, the location of the tuft sensilla including aesthetascs; (2) surgical removal of the aesthetascs and asymmetric sensilla; and (3) surgical removal of the aesthetascs (and some guard and companion setae) but not asymmetric sensilla and nontuft sensilla. However, the flick response to chemical stimuli was also reduced by ablation of nonaesthetasc sensilla on the medial flagella. Therefore we propose that the aesthetasc pathway of odorant processing is necessary for chemically mediated elicitation of the flick response.

The selective removal of either aesthetascs or asymmetric sensilla was a particularly important step in establishing the role of aesthetascs. In a previous study, we reported that odorant-mediated antellular grooming behavior (AGB) was abolished after ablation of aesthetascs and asymmetric sensilla (Wroblewska et al., 2002). Because aesthetascs greatly outnumber the asymmetric sensilla, we concluded that olfactory neurons in the aesthetascs activate AGB. However, Schmidt and Derby (2005) showed through selective ablation of these sensilla that AGB was actually mediated by the asymmetric sensilla. In the present study, we used this finding to verify the effectiveness of the ablation procedure. Ablation of only asymmetric sensilla resulted in loss of AGB, and ablation of only aesthetascs had no effect on AGB. Selective ablation of either asymmetric sensilla or aesthetascs was further confirmed by microscopic examination of the antennules after behavioral assays.

The aesthetascs have also been implicated in other chemically mediated behaviors, in particular those with social context. The aesthetasc pathway alone provides the chemosensory input necessary to recognize and respond to urine cues from shelters occupied by conspecifics (Horner et al., 2008a). The aesthetascs are believed to provide the sole chemosensory input modulating pheromone-mediated courtship behavior in Callinectes sapidus (Gleeson et al., 1982), individual recognition in Homarus americanus (Johnson and Atema, 2005), and social recognition in Procambarus clarkii (Horner et al., 2008b). In these cases, ablation of the aesthetascs resulted in complete loss of the behavior studied. However, the role of other sensilla cannot be definitively excluded in two of these behaviors. According to Gleeson (1982), the only evidence that sensilla on the medial flagella do not contribute to courtship behavior is from a Ph.D. dissertation based on another species of crab, Portunis sanguinolentus (Christofferson, 1970). Ablation of the medial flagella had no effect on individual recognition in H. americanus (Atema et al., 1999). However, the ablation technique employed by Johnson and Atema (2005) removed asymmetric sensilla as well as aesthetascs. Aesthetascs alone are sufficient to allow normal discrimination of odorants, search behavior, and food localization in P. argus, but these behaviors can be as readily performed in the absence of aesthetascs as long as nonaesthetasc sensilla are intact (Steullet et al., 2001, 2002; Horner et al., 2004).

DW ablation of the medial flagella, which contain only nonaesthetasc sensilla, resulted in a significant reduction, but not elimination, of chemically mediated flick response. Six types of nonaesthetasc setae—hooded, plumose, short setuled, long simple, medium simple, and short simple—are scattered along the lengths of the medial flagella and the nontuft regions of the lateral flagella (Cate and Derby, 2001). The medial flagella contain 64% to 80% of these setal types. Thus a sizable population of bimodal setae located on the lateral flagella remained functional after ablation of the medial flagella in addition to the aesthetascs necessary to elicit the behavior. Therefore we propose that the nonaesthetasc pathway of odorant processing is necessary but not sufficient for modulating antennular flicking behavior toward chemical stimuli. If this hypothesis is true, ablation of all nonaesthetasc sensilla in the manner described by Horner et al. (2004) and Steullet et al. (2002) should entirely eliminate chemically mediated flicking. Our results contradict to some degree an earlier study on crayfish that showed that application of odorant to either the medial or lateral flagella alone elicited activity in the depressor muscle driving flicking (Mellon, 1997), which would suggest that nonaesthetasc sensilla are sufficient to modulate the behavior. It is possible that the odorant stimuli applied to the crayfish flagella were of sufficiently high concentration to elicit flicking in both flagella. Or it could be that the nonaesthetasc pathway in crayfish makes a greater contribution to eliciting flicking than it does in lobsters. Crayfish have lower numbers of aesthetascs per flagellum annulus (2–6 vs. 8–10 for P. argus) and fewer olfactory receptor neurons per aesthetasc (40–100 vs. 350 for P. argus) (Tierney et al., 1986; Grünert and Ache, 1988; Mellon et al., 1989).

The nonaesthetasc setae in the tuft region—asymmetric sensilla, guard and companion setae—do not play a role in chemical mediation of antennular flicking, because surgical ablation of these had no effect. Asymmetric sensilla have also been shown not to mediate courtship behavior in C. sapidus (Gleeson, 1982). Therefore, not only are asymmetric sensilla solely responsible for modulating chemosensory mediation of AGB, but AGB is the only one of three behaviors tested directly that is associated with these sensilla (Schmidt and Derby, 2005). No behavior has been linked to activation of either guard or companion setae, even though they are known to be innervated, possibly with mechanosensory neurons (Laverack, 1964).

In summary, we propose that odorant mediation of flicking behavior requires both the aesthetasc and nonaesthetasc pathways. Results of ablation of the aesthetascs show that the nonaesthetasc pathway alone is not sufficient to elicit flicking in response to odorant. Conversely, partial ablation of nonaesthetascs results in a significant attenuation in response by aesthetascs and remaining nonaesthetasc sensilla, a finding that suggests that the aesthetasc pathway alone is also not sufficient to elicit flicking. In contrast, for the behaviors of food localization, search, and odorant discrimination, either pathway is sufficient (Horner et al., 2004; Steullet et al., 2001, 2002).

Unlike search behavior and food localization, which are behaviors also used to assess odorant discrimination (Steullet et al., 2002), flicking is a stereotypic, solely antennule-based behavior. The lateral antennular neropil (LAN) is the specific region of the brain that provides motor output driving flicking behavior, and it receives chemosensory input directly from nonaesthetasc sensilla (Schmidt et al., 1992; Schmidt and Ache, 1996a). Therefore it was our hypothesis before conducting this study that odorant-induced flicking would be a reflexive behavior similar to AGB (Schmidt and Derby, 2005), stimulated by input through the nonaesthetasc pathway. However, our findings show that the nonaesthetasc pathway does not appear to be enough to modulate flicking behavior in response to chemical stimuli; it also requires input from the aesthetascs via the aesthetasc pathway, which does not terminate on the LAN (Schmidt and Ache, 1992, 1996b). Instead, input from the aesthetascs must affect the LAN indirectly, perhaps through local interneurons between the olfactory lobe (OL) and the LAN (Mellon and Alones, 1995; Mellon, 1996; Schmidt et al., 1992; Schmidt and Ache, 1996b).

In discussing the function of antennular flicking in light of the results of the present study, it is important to consider the role of other sensory modalities. Indeed, in crayfish, hydrodynamic and mechanical stimuli applied to either flagellum of the antennules, as well as to the second antennae and the cephalothorax, consistently elicit flicking in crayfish (Mellon, 1997). Our study was designed to consider only chemosensory input via the antennules; odorant stimuli were injected into a continuous stream of tank water supplied to the general region of the antennules through a headset. The very reason for the design of this stimulus delivery system was based on earlier observations that flick rates increased to such a degree through pulsed presentation of stimuli that it was impossible to measure responses to chemical stimuli alone without removing the confounding effect of hydrodynamic (and possibly visual) stimuli (Daniel and Derby, 1991). In nature, proximity of an odorant source such as food or conspecifics will likely be associated with both chemical and hydrodynamic cues. The combination of these cues might be enough to increase flick rate through stimulation of the bimodal nonaesthetasc sensilla alone. However, the greatest flick rates will be attained when both nonaesthetasc and aesthetasc sensilla are stimulated.

Electrophysiological recordings from the local interneurons between the OL and the LAN indicate that they may integrate odorant and hydrodynamic information relevant to antennular movements (Mellon and Alones, 1995; Schmidt and Ache, 1996b; Mellon, 2005, 2007; Humphrey and Mellon, 2007; Mellon and Humphrey, 2007). We suggest that, when activated, the aesthetasc pathway serves to amplify the input from the nonaesthetasc pathway, leading to a maximum increase in flick rate. Thus flicking is most likely to increase when significant levels of odorant are encountered by the aesthetascs on the lateral flagella.

Perhaps the sensory systems modulating flicking are analogous to the organization of photoreceptors in the retina of vertebrates. The retina of many vertebrates contains the area centralis, a small central region containing a particularly high concentration of photoreceptors. Images of interest detected by the less photoreceptor-rich region of the retina outside the area centralis can be projected through eye movements on the area centralis in order to enhance visual acuity. In flicking, a large area—the antennules, the second antennae, and the cephalothorax—serves as the "retina," providing multimodal sensory input modulating the behavior. The size of this area is large enough to greatly increase the probability of encountering odorant patches. However, with the exception of the tuft region of the lateral flagella, receptor density in these regions is low, presumably limiting their acuity. The tuft region, with its high density of aesthetascs, serves as the "area centralis." This is where maximum acuity can be attained if patches of odorant can be "focused" there, perhaps via movements of the antennules or body. The resultant enhanced flicking rate will serve to further increase chemosensory acuity.


   Acknowledgments
 
We thank the staff at Keys Marine Laboratory, Long Key, Florida, for supplying lobsters, and Drs. Charles Derby, Amy Horner, and Manfred Schmidt for fruitful discussions and advice on excision techniques. We also wish to thank the two reviewers of the original manuscript for their very helpful comments.


   Footnotes
 
Received 30 November 2007; accepted 18 March 2008.


   Literature Cited
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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