Identification of chemosensory sensilla mediating antennular flicking behavior in Panulirus argus, the Caribbean spiny lobster.
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; Grunert 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 OMITTED]
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
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 [degrees]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 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.
Table 1 Protocols for ablation techniques and behavioral assays Control (sham) Experiment Procedure Assay DW Ablation Experiment 1 Flagella soaked in ASW Flick assay (sham) Experiment 2 Flagella soaked in ASW Flick assay (sham) Excision Experiments: Part 1 Experiment 1 Lobsters restrained Flick assay (sham) Experiment 2 Lobsters restrained Flick assay (sham) Excision Experiments: Part 1 Experiment 1 GS & CS on lateral Flick & boundary excised AGB (sham) assays Experiment 2 GS & CS on medial Flick & boundary excised AGB (sham) assays First ablation Experiment Procedure Assay DW Ablation Experiment 1 Medial flagella soaked Flick assay in DW Experiment 2 Lateral flagella soaked Flick assay in DW Excision Experiments: Part 1 Experiment 1 GS & CS excised Flick assay Experiment 2 Aesthetascs & Flick assay asymmetric sensilla excised Excision Experiments: Part 1 Experiment 1 Asymmetric sensilla Flick & AGB excised assays Experiment 2 Aesthetascs excised Flick & AGB assays Second ablation Experiment Procedure Assay DW Ablation Experiment 1 Both flagella Flick assay soaked in DW Experiment 2 Excision Experiments: Part 1 Experiment 1 Remaining tuft Flick assay setae excised Experiment 2 NA NA Excision Experiments: Part 1 Experiment 1 NA NA Experiment 2 NA NA AGB, antellular grooming behavior; ASW, artificial seawater; CS, companion setae; DW, distilled water; GS, guard setae; NA, not applicable.
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.
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 [mu]m). This resulted in a 10 mmo[11.sup.-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[degrees]C until needed. Stock solutions of L-Glu (10mmo[11.sup.-1], pH 7.9) were prepared in ASW. The SE ultra-filtrate and L-Glu stock solutions were stored in aliquot samples at -80[degrees]C until needed. On the day of an experiment, odorant solutions were thawed and diluted with ASW to 1 mmo[11.sup.-1] (SE) or 0.5 mmo[11.sup.-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.sup.-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 OMITTED]
[FIGURE 4 OMITTED]
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 [+ or -] 0.16 (mean [+ or -] standard deviation) Hz toward ASW and 0.54 [+ or -] 0.32 Hz toward squid extract (SE). The wipe rate for sham-ablated lobsters, pooled for the second excision experiment, was 1.0 [+ or -] 1.9 toward ASW and 15 [+ or -] 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 OMITTED]
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% [+ or -] 0.424% (mean [+ or -] 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% [+ or -] 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% [+ or -] 5.85% (mean [+ or -] 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% [+ or -] 1.03% of asymmetric sensilla and 13% [+ or -] 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% [+ or -] 6.35% loss of asymmetric sensilla and 97.3% [+ or -] 0.577% loss of aesthetascs (see Fig. 1C, for example).
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; Grunert 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.
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.
Atema, J., T. Breithaupt, A. LeVay, J. Morrison, and M. Edattukaran. 1999. Urine pheromones in the lobster. Homarus americanus: both males and females recognize individuals and only use the lateral antennules for this task. Chem. Senses 24: 615-616.
Barbato, J. C., and P. C. Daniel. 1997. Chemosensory activation of an antennular grooming behavior in the spiny lobster. Panulirus argus, is tuned narrowly to L-glutamate. Biol. Bull. 193: 107-115.
Carr, W. E. S., J. C. Netherton III, R. A. Gleeson, and C. D. Derby. 1996. Stimulants of feeding behavior in fish: analyses of tissues of diverse marine organisms. Biol. Bull. 190: 149-160.
Cate, H.S., and C. D. Derby. 2001. Morphology and distribution of setae on the antennules of the Caribbean spiny lobster Panulirus argus reveal new types of bimodal chemo-mechanosensilla. Cell Tissue Res. 304: 439-454.
Cavanaugh, G. M. 1964. Formulae and Methods. Vol. 5. Marine Biological Laboratory, Woods Hole, MA.
Christofferson, J.P. 1970. An electrophysiological and chemical investigation of the female sex pheromone of the crab Portunis sanguinolentus (Herbst). Ph.D. dissertation, University of Hawaii. Manoa.
Daniel, P.C., and C. D. Derby 1991. Mixture suppression in behavior: the antennular flick response in the spiny lobster towards binary odorant mixtures. Physiol. Behav. 49: 591-601.
Derby, C. D., and J. Atema. 1982. The function of chemo-and mechanoreceptors in lobster (Homarus americanus) feeding behaviour. J. Exp. Biol. 98: 317-328.
Giri, T., and D.W. Dunham. 1999. Use of the inner antennule ramus in the localization of distant food odors by Procambarus clarkii (Girard, 1852) (Decapoda, Cambaridae). Crustaceana 72: 123-127.
Giri, T., and D. W. Dunham. 2000. Female crayfish (Procambarus clarkii (Girard, 1852)) use both antennular rami in the localization of male odour. Crustaceana 73: 47-458.
Gleeson, R. A. 1982. Morphological and behavioral identification of the sensory structures mediating pheromone reception in the blue crab, Callinectes sapidus. Biol. Bull. 163: 162-171.
Gleeson, R. A., W. E. S. Carr, and H. G. Trapido-Rosenthal. 1993. Morphological characteristics facilitating stimulus access and removal in the olfactory organ of the spiny lobster, Panulirus argus: insight from the design. Chem. Senses 18: 67-75.
Gleeson, R. A., L. M. McDowell, and H. C. Aldrich, 1996. Structure of the aesthetasc (olfactory) sensilla of the blue crab, Callinectes sapidus: transformations as a function of salinity. Cell Tissue Res. 284: 279-288.
Goldman, J. A., and M. A. R. Koehl. 2001. Fluid dynamic design of lobster olfactory organs: high-speed kinemetic analysis of antennule flicking by Panulirus argus. Chem. Senses 26: 385-398.
Grunert, U., and B. W. Ache. 1988. Ultrastructure of the aesthetasc (olfactory) sensilla of the spiny lobster, Panulirus argus. Cell Tissue Res. 251: 95-103.
Harrison, P. J. H., H. S. Cate, P. Steullet, and C. D. Derby. 2001. Structural plasticity in the olfactory system of adult spiny lobsters: postembryonic development permits life-long growth, turnover, and regeneration. Mar. Freshw. Res. 52: 1357-1365.
Horner, A. J., M. J. Weissburg, and C.D. Derby. 2004. Dual antennular chemosensory pathways can mediate orientation by Caribbean spiny lobsters in naturalistic flow conditions. J. Exp. Biol. 207: 3785-3796.
Horner, A. J., M. J. Weissburg, and C. D. Derby. 2008a. The olfactory pathway mediates sheltering behavior of Caribbean spiny lobsters, Panulirus argus, to conspecific signals. J. Comp. Physiol. A 194: 243-253.
Horner, A. J., M. Schmidt, D. H. Edwards, and C. D. Derby. 2008b. Role of the olfactory pathway in agonistic behavior of crayfish, Procambarus clarkii, Invertebr. Neurosci. 8: 11-18.
Humphrey, J. A. C., and DeF. Mellon, Jr. 2007. Analytical and numerical investigation of the flow past the lateral antennular flagellum of the crayfish Procambarus clarkii. J. Exp. Biol. 210: 2969-2978.
Johnson, M. E., and J. Atema. 2005. The olfactory pathway for individual recognition in the American lobster Homarus americanus. J. Exp. Biol. 208: 2865-2872.
Karavanich, C., and J. Atema. 1998. Olfactory recognition of urine signals in dominance fights between male lobsters, Homarus americanus. Behaviour 135: 719-730.
Keller, T. A. I. Powell, and M. J. Weissburg. 2003. Role of olfactory appendages in chemically mediated orientation of blue crabs. Mar. Ecol. Prog. Ser. 216: 217-231.
Koehl, M. A. R. 2006. The fluid mechanics of arthropod sniffing in turbulent odor plumes. Chem. Senses 31: 93-105.
Koehl, M. A. R., J. R. Koseff, J. P. Crimaldi, M. G. McKay, T. Cooper, M. B. Wiley, and P. A. Moore. 2001. Lobster sniffing: antennules design and hydrodynamic filtering of information in an odor plume. Science 294: 1948-1951.
Laverack, M. S. 1964. The antennular sense organs of Panulirus argus. Comp. Biochem. Physiol. 13: 301-321.
Laverack, M. S., and D. J. Ardil. 1965. The innervation of aesthetasc hairs of Panulirus argus. Q. J. Microsc. Sci. 106: 45-60.
Maynard, D. M. 1965. Integration in crustacean ganglia. Symp. Soc. Exp. Biol. 20: 111-149.
Mellon, DeF., Jr. 1996. Dynamic response properties of broad spectrum olfactory interneurons in the crayfish midbrain. Mar. Behav. Physiol. 27: 111-126.
Mellon, DeF., Jr. 1997. Physiological characterization of antennular flicking reflexes in the crayfish. J. Comp. Physiol. A 180: 553-565.
Mellon, DeF., Jr. 2005. Integration of hydrodynamic and odorant inputs by local interneurons of the crayfish deutocerebrurm. Exp. Biol. 208: 3711-3720.
Mellon, DeF., Jr. 2007. Combining dissimilar senses: central processing of hydrodynamic and chemosensory inputs in aquatic crustaceans. Biol. Bull 213:1-11.
Mellon, DeF., Jr., and V. E. Alones. 1995. Identification of three classes of multiglomerular, broad-spectrum neurons in the crayfish olfactory mid-brain by correlated patterns of electrical activity and dendritic arborization. J. Comp. Physiol. A 177: 55-71.
Mellon, DeF., Jr., and J. A. C. Humphrey. 2007. Directional asymmetry in the response of local interneurons in the crayfish deutocerebrurm to hydrodynamic stimulation of the lateral antennular flagellum. J. Exp. Biol. 210: 2961-2968.
Mellon, DeF., Jr., H. R. Tuten, and J. Redick, 1989. Distribution of radioactive leucine following uptake by olfactory sensory neurons in normal and heteromorphic antennules. J. Comp. Neurol. 280: 645-667.
Price, R. B., and B. W. Ache. 1977. Peripheral modification of chemosensory information in the spiny lobster. Comp. Biochem. Physiol. A 57: 249-253.
Reeder, P. B., and B. W. Ache. 1980. Chemotaxis in the Florida lobster, Panulirus argus. Anim. Behav. 28: 831-839.
Roye, D. B. 1994. Antennular withdrawal motoneurons in the lateral antennular neuropil of Callinectes sapidus. J. Crustac. Biol. 14: 484-496.
Schmidt, M., and B. W. Ache. 1992. Antennular projections to the midbrain of the spiny lobster. II. Sensory innervations of the olfactory lobe. J. Comp. Neurol. 318: 291-303.
Schmidt, M., and B. W. Ache. 1993. Antennular projections to the midbrain of the spiny lobster. III. Central arborizations of motoneurons. J. Comp. Neurol. 336: 583-594.
Schmidt, M., and B. W. Ache. 1996a. Processing of antennular input in the brain of the spiny lobster, Panulirus argus. I. Non-olfactory chemosensory and mechanosensory pathway of the lateral and median antennular neuropils. J. Comp. Physiol. A 178: 579-604.
Schmidt, M., and B. W. Ache. 1996b. Processing of antennular input in the brain of the spiny lobster, Panulirus argus. II. The olfactory pathway. J. Comp. Physiol. A 178: 605-628.
Schmitt, B. C., and B. W. Ache. 1979. Olfaction: responses of a decapod crustacean are enhanced by flicking. Science 205: 204-206.
Schmidt, M., and C. D. Derby. 2005. Non-olfactory chemoreceptors in asymmetric setae activate antennular grooming behavior in the Caribbean spiny lobster Panulirus argus. J. Exp. Biol. 208: 233-248.
Schmidt, M., L. Van Ekeris, and B. W. Ache. 1992. Antennular projections to the midbrain of the spiny lobster. I. Sensory innervations of the lateral and medial neuropils. J. Comp. Neurol. 318: 277-290.
Snow, P. J. 1973. The antennular activities of the hermit crab, Pagurus alaskensis (Benedikt). J. Exp. Biol. 58: 745-768.
Spencer, M., and K. A. Linberg. 1986. Ultrastructure of the aesthetasc innervation and external morphology of the lateral antennule setae of the spiny lobster Panulirus interruptus (Randall). Cell Tissue Res. 245: 69-80.
Steullet, P., H. S. Cate, and C. D. Derby. 2000. A spatiotemporal wave of turnover and functional maturation ptor neurons in the spiny lobster, Panulirus argus. J. Neurosci. 20: 3282-3294.
Steullet, P., O. Dudar, T. Flavus, M. Zhou, and C. D. Derby. 2001. Selective ablation of antennular sensilla on the Caribbean spiny lobster Panulirus argus suggests that dual antennular chemosensory pathways mediate odorant activation of searching and localization of food. J. Exp. Biol. 204: 4259-4269.
Steullet, P., D. R. Krutzfeldt, G. Hamidani, T. Flavus, V. Ngo, and C. D. Derby. 2002. Dual antennular chemosensory pathways mediate odor-associative learning and odor discrimination in the Caribbean spiny lobster Panulirus argus. J. Exp. Biol. 205: 851-867.
Sullivan, J. M., and B. S. Beltz. 2001. Development and connectivity of olfactory pathways in the brain of the lobster Homarus americanus. J. Comp. Neurol. 441: 23-43.
Tierney, A. J., C. S. Thompson, and D. W. Dunham. 1986. Fine structure of aesthetasc chemoreceptors in the crayfish Orconectes propinquus. Can. J. Zool. 64: 392-399.
Wroblewska, J., S. Whalley, M. Fischetti, and P. C. Daniel. 2002. Identification of chemosensory sensilla activating antennular grooming behavior in the Caribbean spiny lobster, Panulirus argus. Chem. Senses 27: 769-778.
Received 30 November 2007; accepted 18 March 2008.
* To whom correspondence should be addressed. E-mail: email@example.com
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.
PETER C.DANIEL (1), *, MICHAEL FOX (2), AND SAGAR MEHTA (3)
(1) Department of Biology, Hofstra University, Hempstead, New York 11549; (2) Plainview Old Bethpage John F. Kennedy High School, Plainview, New York 11803; and (3) The Wheatley School, Old Westbury, New York 11568
|Printer friendly Cite/link Email Feedback|
|Author:||Daniel, Peter C.; Fox, Michael; Mehta, Sagar|
|Publication:||The Biological Bulletin|
|Date:||Aug 1, 2008|
|Previous Article:||Evolutionary and structural diversification of the larval nervous system among marine bryozoans.|
|Next Article:||Rhythms of locomotion expressed by Limulus polyphemus, the American horseshoe crab: I. Synchronization by artificial tides.|