Dual antennular chemosensory pathways can mediate orientation by Caribbean spiny lobsters in naturalistic flow conditions
1 Department of Biology, Georgia State University, PO Box 4010, Atlanta, GA
30302-4010, USA
2 School of Biology, Georgia Institute of Technology, 311 Ferst Drive,
Atlanta, GA 30332, USA
* Author for correspondence (e-mail: bioajh{at}langate.gsu.edu)
Accepted 19 July 2004
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Summary |
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Key words: olfaction, Crustacea, aesthetasc, odor, flume, Panulirus argus
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Introduction |
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Chemical stimuli are detected by a multitude of chemoreceptive structures
on crustaceans. Although chemoreceptive sensilla can be found on virtually all
body surfaces, they are most concentrated on the appendages, particularly the
antennules, antennae, dactyls and mouthparts
(Ache and Macmillan, 1980;
Derby, 1982
;
Schmidt, 1989
;
Schmidt and Gnatzy, 1984
; Cate
and Derby, 2001
,
2002a
;
Garm et al., 2003
). The
antennules in particular have long been considered to be the primary
chemoreceptive organ of the spiny lobster
(Fig. 1). Each antennule is
composed of four segments, the most distal of which bifurcates into a lateral
flagellum and a medial flagellum. Each flagellum is composed of annuli that
bear a complement of chemo- and mechanosensory sensilla that vary in
morphology, distribution and pattern of innervation. Many studies have shown
that the antennules are important for distance chemoreception in lobsters
(Reeder and Ache, 1980
;
Devine and Atema, 1982
) and
other decapod crustaceans (Hazlett,
1971a
; Kraus-Epley and Moore,
2002
); however, it is not clear which populations of antennular
sensilla are involved in this behavior.
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Chemosensory information from the antennular sensilla is transmitted to the
central nervous system in two parallel pathways: the aesthetasc/olfactory lobe
pathway and the non-aesthetasc/lateral antennular neuropil pathway (Schmidt
and Ache, 1992,
1996a
,b
;
Schmidt et al., 1992
). The
aesthetasc/olfactory lobe pathway originates in clusters of olfactory receptor
neurons innervating the prominent aesthetasc sensilla. Aesthetascs are the
most numerous sensilla on the antennules of the Caribbean spiny lobster,
Panulirus argus, and are located exclusively in a distal tuft on the
ventral face of each lateral flagellum. Aesthetascs are unique among
antennular sensilla characterized thus far because they are innervated
exclusively by chemosensory neurons. Each aesthetasc is innervated by the
dendrites of approximately 300 olfactory receptor neurons
(Grünert and Ache, 1988
;
Steullet et al., 2000
;
Derby et al., 2003
), whose
axons project to glomeruli within the paired olfactory lobes of the brain
(Schmidt et al., 1992
;
Schmidt and Ache, 1996b
;
Sandeman and Mellon, 2002
).
Aesthetascs were traditionally believed to be the most important structures
for detecting, discriminating and localizing odors because of their great
numbers and extensive innervation by chemosensory neurons. Indeed, several
studies have shown that ablation of the lateral flagellum impairs
odor-mediated behaviors (Devine and Atema,
1982
; Reeder and Ache,
1980
; Giri and Dunham,
1999
; Kraus-Epley and Moore,
2002
; Wroblewska et al.,
2002
). These behavioral impairments were often attributed
exclusively to the loss of aesthetasc sensilla because they are the most
numerous sensillar type on the lateral flagellum. However, more recent work
has shown that the aesthetascs are not the only structures on the antennule
capable of driving food-odor-mediated behaviors
(Derby et al., 2001
; Steullet
et al., 2001
,
2002
).
Nine other types of sensilla, collectively referred to as
`non-aesthetascs', are widely distributed on the antennules of P.
argus, and at least four of these (hooded, long simple, medium simple and
asymmetric sensilla) are bimodal and innervated by distinct populations of
chemoreceptive and mechanoreceptive neurons (Cate and Derby,
2001,
2002b
;
Schmidt et al., 2003
).
Backfills of the antennular nerve have revealed that presumptive chemo- and
mechanosensory neurons innervating non-aesthetasc sensilla on the antennular
flagella project to the stratified lateral antennular neuropils, while those
on the proximal segments and statocysts project to the unstructured median
antennular neuropil (Schmidt et al.,
1992
; Schmidt and Ache,
1993
,
1996a
;
Cate and Roye, 1997
), thus
forming the non-aesthetasc chemosensory pathway.
Furthermore, these pathways remain anatomically distinct at the next
synaptic pathway. Output interneurons from the olfactory lobes and from the
lateral antennular neuropil project to different regions of the terminal
medullae (Sullivan and Beltz,
2001). It should be noted, however, that there is some
connectivity between these two neuropils; for example, local olfactory
interneurons exist that connect the ipsilateral olfactory lobe and lateral
antennular neuropil (Mellon and Alones,
1994
; Schmidt and Ache,
1996b
).
Although the two pathways have distinct anatomical arrangements, the
functional significance of this dual organization remains unclear. To date, no
published studies have conclusively demonstrated unique functions in
food-odor-mediated behaviors for either pathway in spiny lobsters. In fact,
previous work has generally found an overlap in the functions of the
aesthetasc and non-aesthetasc pathways for behaviors such as odorant
activation of searching behavior, odor learning and discrimination of food
odors in small-scale, low-flow arenas (Steullet et al.,
2001,
2002
).
The importance of each pathway for behaviors over a larger spatial scale in
more complex flows, such as those occurring during orientation to distant
food-odor stimuli, has not been as thoroughly studied. Odor plumes emanating
from sources in realistic flow conditions are spatially and temporally complex
(Webster and Weissburg, 2001),
and perhaps extracting orientational information from these signals requires a
specialized neural pathway. Previous studies examining orientation behavior in
flumes have focused more on uncovering the organism's method of orientation
(e.g. tropotaxis, odor-gated rheotaxis) or on the role of entire antennular
flagella than on determining the specific sensilla or chemosensory pathways
involved in orientation (McLeese,
1973
; Reeder and Ache,
1980
; Devine and Atema,
1982
; Atema, 1995
;
Beglane et al., 1997
;
Weissburg, 2000
;
Kozlowski et al., 2001
). In
several of these studies, entire flagella (including both aesthetasc and
non-aesthetasc sensilla) were ablated, while in others, ablations were only
performed unilaterally. Because the ablations were not specific to a single
population of sensilla, the importance of each pathway for orientation remains
unknown.
Therefore, the goal of this work is to determine whether the aesthetasc pathway or the non-aesthetasc pathway is necessary and sufficient for locating the source of a distant food odor stimulus. To assess the importance of each pathway for this task, we systematically ablated different populations of antennular sensilla and compared the behavior of ablated animals to that of intact controls. Under the conditions tested, both the aesthetasc and non-aesthetasc pathways were sufficient for orientation, but neither pathway alone was necessary. Overall, the results suggest that there is an overlap in the function of the pathways and that food searching is not a unique function of either pathway alone.
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Materials and methods |
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Ablations
To assess the importance of different populations of antennular sensilla
for orientation, we performed four bilateral ablations, which are described
below and summarized in Table
1. The four ablations have been used previously, and their
effectiveness has been confirmed both morphologically and
electrophysiologically (Steullet et al.,
2001,
2002
). All ablations were
performed on non-anesthetized spiny lobsters immobilized on a plastic
restraining device within a shallow container of artificial seawater.
Ablations requiring surgical removal of sensilla were performed once, at least
three days prior to the start of a series of experimental trials, using a
hand-tooled narrow blade (0.2 mm wide;
Steullet et al., 2001
).
Chemical ablations were performed with distilled water within 24 h of the
start of each trial. At the conclusion of each series of experimental trials,
ablated antennules were excised and the efficacy of ablation was evaluated
using light microscopy to count the number of sensilla that remained intact on
each antennule. This analysis confirmed that shaving was a highly reliable
method for removing sensilla. Shaving removed 99.8±0.04% (mean ±
S.E.M., N=13) of all aesthetascs on
the antennule, which is similar to values obtained in other studies
(Steullet et al., 2001
). This
corresponds to 12 intact aesthetascs per animal for the animals that we
used in this study.
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Control
Control animals were immobilized in the plastic restraining device in the
same manner as ablated animals, but no sensilla were removed or
inactivated.
All antennular flagellar chemoreceptors ablated
Aesthetasc and non-aesthetasc chemosensory neurons on both antennules were
chemically ablated by immersing the lateral and medial flagella of each
antennule in a tube of distilled water for 15 min. Distilled water
functionally inactivates chemosensory neurons by disrupting the osmotic
balance of the outer dendrites (Derby and
Atema, 1982; Gleeson et al.,
1997
). The ablation is temporary and reversible, lasting only
24 h before the neurons once again respond to chemical stimuli
(Derby and Atema, 1982
;
Steullet et al., 2001
).
Because of the ephemeral nature of this ablation, it was performed within 24 h
of each experimental trial. Distilled water effectively inactivates
chemosensory neurons but may also affect the function of some types of
mechanosensory neurons. Mechanosensory neurons with dendrites projecting up
the length of the sensillum may be exposed to and inactivated by the distilled
water environment (Derby and Atema,
1982
; Garm et al., in
press
).
Aesthetascs ablated
All aesthetasc sensilla on both lateral flagella were surgically removed at
the base using a hand-tooled blade. Asymmetric setae, which are located
laterally to the aesthetasc rows (Gleeson
et al., 1993; Cate and Derby,
2001
), were also removed during this ablation. Removal of
aesthetascs in this manner obliterates the chemosensory dendrites of the
sensillum, which results first in unresponsiveness to odors, followed by death
and degradation of the receptor neurons
(Harrison et al., 2001
).
Non-aesthetasc chemo- and mechanoreceptors ablated
All visible non-aesthetasc sensilla were surgically removed from the entire
length of the lateral and medial flagella of both antennules. The flagella
were then coated with a thin layer of cyanoacrylate glue (Super Glue Corp.,
Rancho Cucamonga, CA, USA) to prevent stimulus access to any remaining, unseen
non-aesthetasc sensilla. Covering the antennules with cyanoacrylate glue
effectively prevents stimulation of both non-aesthetasc chemosensory neurons
and mechanosensory neurons that are responsive to hydrodynamic and some
tactile stimuli (Derby and Atema,
1982).
Non-aesthetasc chemoreceptors ablated
This ablation was designed to specifically eliminate the function of
non-aesthetasc chemoreceptors while maintaining the integrity of at least some
non-aesthetasc mechanoreceptors. Non-aesthetasc sensilla were surgically
removed from annuli located within the aesthetasc region of each lateral
flagellum. The shaved region was then coated with a thin layer of
cyanoacrylate glue to prevent stimulus access to any remaining non-aesthetasc
sensilla. The rest of the antennule (medial flagellum and proximal region of
lateral flagellum) was then immersed in distilled water for 15 min to ablate
non-aesthetasc chemoreceptor neurons in these regions. The aesthetasc region
on each lateral flagellum was maintained in seawater during this process.
Although the shaving and gluing inactivated mechanoreceptor neurons within the
aesthetasc region, at least some of the mechanoreceptors along the medial
flagellum and proximal portion of the lateral flagellum probably remained
intact and functional (see above All antennular flagellar
chemoreceptors ablated for explanation).
Odor stimuli
Three different odor stimuli were used in the experiments. Control stimuli
consisted of artificial seawater (Instant Ocean®) taken directly from the
flume before the start of trials, and experimental stimuli consisted of two
concentrations of shrimp extract. Shrimp extract is a potent feeding stimulus
for spiny lobsters (Carr, 1988;
Derby, 2000
) and was prepared
by homogenizing frozen shrimp in artificial seawater with a blender and then
collecting and freezing the raw extract in 10-ml aliquots. The final
concentration of the raw extract was approximately 300 g l1.
We then made dilutions of this stimulus by mixing raw shrimp extract in
artificial seawater taken directly from the flume. Each stimulus was
thoroughly mixed by shaking and filtered through Whatman #5 filter paper to
remove large pieces of shrimp material. Preliminary experiments showed that
shrimp concentrations of 3 and 0.3 g l1 were effective in
attracting lobsters to the odor source, so they were used in subsequent trials
and were called `high' and `low' concentrations, respectively.
Experimental setup
To simulate semi-natural flow conditions where fluid flow and boundary
layer conditions could be controlled, all trials were conducted in a
recirculating 5000-liter flume housed at Georgia Institute of Technology
(Fig. 2). (See
Webster and Weissburg, 2001;
Weissburg et al., 2003
;
Keller et al., 2003
for
descriptions of the flume and its use in examining chemosensory behavior of
other animals.) The flume measured 12.5 m long, 0.75 m wide and 0.35 m high,
and the 2 m working section for this study began 10 m downstream of the entry
way and ended 0.5 m upstream of the reservoir
(Fig. 2). The floor of the
flume was covered with a 1 cm-deep layer of fine-grained quartz sand, and the
side walls were covered with black panels to eliminate confounding visual
cues. The flume was filled with artificial seawater (Instant Ocean®) at
22°C. Flow velocity was 4.9±0.08 cm s1 (mean
± S.D.), as measured by an acoustic-Doppler flow
meter, with a water depth of 23.0±0.348 cm (mean ±
S.D.) controlled by a vertical tailgate. At this flow
speed, the boundary layer shear velocity, u*, calculated using the
law-of-the-wall equation, and boundary layer structure conformed well to
expectations for turbulence in open channel flows
(Keller et al., 2003
). The
near-bed flow was smooth (Reynold's number = 2.65) with a u* of 3.1
mm s1. A cage (0.32 m long x 0.31 m wide x 0.18
m high) constructed out of plastic grating (1x1 cm grate size) was
placed at the downstream end of the working section. Odor stimuli were
released parallel to the flow 2 m upstream from the cage and 2.5 cm above the
bed from a 4.7 mm-diameter brass nozzle with a fairing to minimize the flow
perturbation. Control and experimental odors were introduced into the flume by
a peristaltic pump, which pushed the stimuli through the nozzle at
approximately the same velocity as the background flow (i.e. isokinetic
release of the stimulus).
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All trials were conducted during the day under low light conditions.
Although P. argus is nocturnal in the natural environment, in the
laboratory spiny lobsters will search when presented with food odors during
the day if light levels are low enough. A video camera mounted above the flume
was used to track the two-dimensional movements of the animals. Prior to the
start of a trial, each animal was fitted with a watertight silicone (Sylgard)
backpack containing two red light emitting diodes to facilitate tracking
(Weissburg et al., 2002;
Keller et al., 2003
). The
backpack was attached to the animal by a strip of Velcro® that had been
glued to the carapace. The presence of the backpack had no apparent effect on
the behavior of the animal.
Fifteen minutes before the start of each trial, the lobster was fitted with the backpack and placed in the cage. This was done in order to acclimate the animal to the flume conditions and to provide a constant starting point for each trial. At the end of this acclimation period, the odor stimulus was introduced into the flow, and 30 s later the cage door was opened, allowing the animal to exit and move freely around the flume. The task was for the animal to exit the cage, track the odor plume to its source and physically grab the nozzle.
Each trial lasted a maximum of 10 min. Each spiny lobster had 5 min to completely exit the cage. If the animal did not exit the cage within 5 min, the trial was terminated immediately. If the animal did exit the cage within the first 5 min, it was then given an additional 5 min to locate the odor source and grab the nozzle. A trial ended either when the animal successfully located the odor source and held onto the nozzle or when the additional 5-min period had expired. Lobsters were offered a piece of shrimp at the conclusion of every trial as a test of motivational state. The lobster was removed from the experiments and was not included in the final data set if it failed to take the shrimp. This was done to ensure that an unsuccessful search attempt was due to sensory deficits rather than lack of interest in food.
Each animal was tested a total of three times over the course of 3 days (once each day in one of the three stimulus concentrations). The order of stimulus presentation was randomly determined for each animal prior to the start of the experiment. The three trials were not necessarily run on consecutive days, but all were conducted within a two-week period so that no animal was housed at Georgia Institute of Technology for more than two weeks.
Immobilization of second antennae
When spiny lobsters search for the source of an odor stimulus, they
typically walk with their second antennae positioned perpendicularly to the
long axis of the body. During the course of our experiments, we observed that
some lobsters walked towards the source with one antenna in constant contact
with the side wall of the flume. We were concerned that this additional
contact might enhance search efficiency and mask any possible deficits caused
by antennular ablations. To identify any possible confounding effects of
physical contact between the flume walls and second antennae, we conducted a
series of trials using animals with and without their second antennae
immobilized. We chose to immobilize rather than remove the second antennae
because immobilization was a less severe treatment that retains some sensory
function of the antennae and limits non-specific effects. The second antennae
of five lobsters were positioned above and parallel to the long axis of the
body and secured in this position by binding the two antennae together and
then to the horns above each eye with plastic-coated wire. This arrangement
restricted the movement of the second antennae and thus prevented the animals
from extending them perpendicularly from the body. If the animals were relying
on physical contact with the wall to move towards the source, then we would
expect that those with restrained antennae would have less direct search paths
because they would have to move further from the center of the flume in order
to bring their antennae in contact with the wall.
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Results |
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In contrast to control animals, animals with all antennular flagella
chemoreceptors ablated generally did not locate the odor source when exposed
to either of the shrimp extract stimuli or the seawater control
(Fig. 3). Post-test feeding
responses to shrimp showed that the lack of response in this group of animals
was not due to low motivation. Less than 16% of the animals in this treatment
group failed to respond to the post-test shrimp. Similar response levels were
observed with the other treatment groups, and there was no difference in the
percentage of animals that did not respond to the post-test shrimp between the
five treatment groups [20.05,4=5.48001;
P>0.05]. Thus, functional antennular chemosensilla are necessary
for locating the odor source. However, neither the aesthetascs alone nor the
non-aesthetascs alone are required to mediate this behavior.
Aesthetasc-ablated animals responded similarly to control animals (Fig.3) and there were no significant differences between the percentage of control versus aesthetasc-ablated animals finding the source. Aesthetasc-ablated animals found the source frequently when high (77%; N=13) and low (69%; N=13) concentrations of shrimp extract were used as an odorant and rarely left the cage, let alone found the source, when seawater was used as an odorant. Thus, aesthetascs alone are not necessary, and non-aesthetascs alone are sufficient to drive this behavior.
Non-aesthetasc chemo- and mechanoreceptor-ablated animals also responded similarly to the control animals (Fig. 3). They also located the source regularly in response to both the high (64%, N=11) and low (45%, N=11) concentrations of shrimp stimulus and they did not locate the source with seawater. The success rate of non-aesthetasc chemoreceptor-ablated animals was not significantly different from that of the non-aesthetasc chemo- and mechanoreceptor-ablated animals (Fig.3), although there were fewer animals in this treatment (N=8).
The combination of these results suggests that antennular flagellar
chemoreceptors are necessary for spiny lobsters to locate an odor source but
that either the aesthetascs alone or the non-aesthetascs alone are sufficient
to accomplish this task. Additionally, over the short time frame of these
experiments, we saw no evidence that non-antennular chemoreceptors may be able
to compensate for the loss of antennular chemoreceptors, as has been shown
over a longer time period for other species
(Hazlett, 1971b).
Search efficiency
In addition to recording the overall success rate of animals in each
treatment group, we also examined the efficiency of successful searches to
identify more subtle influences of the ablations. Search efficiency was
quantified using four parameters that are commonly used in orientation
experiments (Devine and Atema,
1982; Moore et al.,
1991
; Moore and Grills,
1999
; Kraus-Epley and Moore,
2002
; Keller et al.,
2003
). The parameters were mean time to locate the odor source
(Fig. 4A), net-to-gross
displacement ratio (NGDR; Fig.
4B), mean walking speed (Fig.
4C) and mean heading angle with respect to the source
(Fig. 4D). Because our analysis
of efficiency is limited only to successful searches, we did not include
completely ablated animals or searches with seawater as a stimulus (since
animals did not locate the source under these conditions).
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Mean time to locate the source
The mean time to locate the odor source was calculated as the average time
difference between exiting the cage and grabbing the source for all animals in
the treatment group. For animals tested in both the high and low shrimp
stimulus concentrations, the time to locate the odor source was not different
for control and ablated groups (Fig.
4A). All four groups of animals found the source within 96 s in
the high concentration and 176 s in the low concentration
(Fig. 4A).
Net-to-gross displacement ratio
The NGDR was used to describe the directness of a search path. The ratio
was calculated as the Euclidean distance from the cage to the nozzle divided
by the total distance traveled by the animal. Ratios approaching 1 represent
more direct paths to the source whereas values approaching 0 represent
increasingly more tortuous paths to the source. The NGDR values of the control
and the three ablated groups were not different when the animals were tested
in the low concentration of shrimp extract
(Fig. 4B). By contrast, when
tested in the high stimulus concentration, there was a significant difference
in the NGDR between control and ablated groups (aesthetascs ablated,
non-aesthetasc chemo- and mechanoreceptors ablated)
(Fig. 4B). Control animals took
very direct paths to the source (NGDR=0.82; N=14). Compared with
control animals, aesthetasc-ablated and non-aesthetasc chemo- and
mechanoreceptor-ablated animals took significantly more tortuous paths to the
source, with NGDR values of 0.63 (N=10) and 0.62 (N=6),
respectively (Fig. 4B).
Interestingly, although these ablated groups differed from the control group,
they did not significantly differ from one another. Thus, the different
ablations produced a similar deficit in this measure of search efficiency.
Mean walking speed
The mean walking speed for each orientation path was calculated by
averaging the speed of the animal over 1-s intervals. There were no
significant differences between the walking speed of control and ablated
animals in either of the stimulus concentrations
(Fig. 4C). In fact, the mean
walking speed of all the groups remained relatively constant over all trials
regardless of stimuli being tested. Animals tested with seawater as a stimulus
walked at similar speeds (in the range of 36 cm s1)
to those tested with the shrimp extracts.
Heading angle with respect to the odor source
Heading angle with respect to the odor source was determined using the
methodology of Moore et al.
(1991). Heading angle was
calculated as the absolute value of the angle between a straight line
connecting the lobster's current position on the search path (based on the
location of the first light-emitting diode of the backpack) and the nozzle,
and a straight line connecting the lobster's current position on the search
path and the lobster's next position on the search path. Values ranged between
0 and 180°, with 0° heading directly towards the source and 180°
heading directly away from the source. There were no significant differences
between the heading angles of control and ablated animals in either of the
stimulus concentrations (Fig.
4D).
Effects of stimulus concentration
The behavior of the lobsters was somewhat dependent on the concentration of
shrimp odor extract. Animals tended to find the odor source more successfully
in high than in low shrimp concentration (Fisher exact test, P=0.06).
Animals performed more efficient searches in the high compared with the low
concentration of shrimp for two of the behaviors, as suggested by the higher
NGDR and lower heading angles in high vs low plumes
(Fig. 4B, ANOVA,
F1,57=5.42, P=0.023 for NGDR;
Fig. 4D, WatsonWilliams
test, F1,57=6.909, P=0.025 for heading angle).
Additionally, there was a strong trend for animals in the high vs low
plumes to locate the odor source more quickly
(Fig. 4A, ANOVA,
F1,57=3.45, P=0.068) and a weak trend for them to
walk faster (Fig. 4C, ANOVA,
F1,57=1.95, P=0.168).
Effects of mechanical stimulation of the second antennae
There was no difference between the percentages of animals locating the
odor source with free or immobilized antennae
(Fig. 5A). Both groups of
animals found the source regularly when tested with the high concentration of
shrimp extract, and neither group located the source with seawater as an
odorant (Fig. 5A).
Additionally, in all measures of search efficiency (mean time to source, NGDR,
mean walking speed and mean heading angle), the two groups of animals did not
differ (Fig. 5BE). Thus,
contact between the second antennae and the wall of the flume does not
significantly influence the success or efficiency of search in our flume.
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Discussion |
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Either aesthetasc or non-aesthetasc chemosensory neurons can mediate food localization behavior
Several previous studies have demonstrated that distance chemoreception in
decapod crustaceans is mediated primarily by antennular chemoreceptors
(Reeder and Ache, 1980;
Devine and Atema, 1982
;
Hazlett, 1971a
;
Kraus-Epley and Moore, 2002
),
and the results of the present study also support this conclusion. When all
antennular flagellar chemoreceptors were ablated, spiny lobsters lost the
ability to locate the source of a 2 m-distant food odor stimulus
(Fig. 3). They did, however,
respond to a piece of shrimp brought into contact with their legs, indicating
that the impairment was due to sensory deficit rather than lack of motivation
to feed. Thus, chemosensory input from antennular sensilla in general is
necessary for orientation. However, the presence of only a subset of
functional chemoreceptors is sufficient to enable orientation.
Aesthetasc-ablated lobsters were as successful as control animals in locating
the odor source (Fig. 3). The
same pattern of behavior was seen in non-aesthetasc-ablated animals
(Fig. 3). Thus, either of the
two chemosensory pathways the aesthetasc pathway or the non-aesthetasc
pathway is alone sufficient to allow orientation. Although it did not
affect the percentage of animals that successfully located the odor source,
ablation of a single pathway did affect search efficiency in some cases. For
example, when the aesthetasc pathway was ablated, animals took more circuitous
paths to the odor source than control animals in the high shrimp stimulus
condition (Fig.
4B).Interestingly, the same deficit was seen when the
non-aesthetasc pathway alone was ablated
(Fig. 4B), further suggesting
an overlapping role for the pathways in our assay.
The results of this study strongly suggest that the two antennular chemosensory pathways are equally able to mediate the behavior under the current experimental conditions. There were no statistically significant differences in the percentage of animals locating the source (Fig. 3) or in the search efficiency (Fig. 4AD) between aesthetasc-ablated and non-aesthetasc-ablated animals. Thus, the function of the aesthetasc and non-aesthetasc pathways in this behavioral assay appears to overlap. Possible reasons for the observed overlap and potentially unique roles for each pathway in odor-mediated behaviors are discussed in later sections.
Non-antennular sensors and non-odor stimuli are not sufficient to mediate food localization behavior
In addition to the antennules, chemosensilla are concentrated on several
other body regions of the spiny lobster, including the walking legs,
mouthparts and second antennae (Derby and
Atema, 1982; Cate and Derby,
2002a
; Garm et al.,
2003
). Work on other decapod crustaceans has shown that leg
chemoreceptors in particular can aid in orientation as the animal approaches
the source of an odor stimulus (Devine and
Atema, 1982
; Moore et al.,
1991
; Keller et al.,
2003
). In our experiments, however, inputs from chemosensilla on
the legs or other regions of the body were not sufficient to allow the lobster
to overcome the sensory deficits caused by antennular ablation. Spiny lobsters
with all antennular chemoreceptors ablated did not locate the odor source even
though all other non-antennular chemoreceptors were intact
(Fig. 3). Although our results
suggest that non-antennular chemosensory inputs are not sufficient to drive
orientation behavior, they do not suggest that these inputs are unimportant or
unnecessary. In the natural environment, spiny lobsters are likely to use a
combination of receptor inputs to efficiently locate prey in order to avoid
unnecessary exposure to predators.
Additionally, visual, hydrodynamic and tactile cues were not sufficient to allow the lobsters to locate the odor source in the absence of chemical stimulation of the antennules. Spiny lobsters did not locate the nozzle when seawater was used as a stimulus, even though visual and hydrodynamic cues would have been comparable between seawater and shrimp odorant trials. Thus, the lobsters in our study were not simply locating the nozzle by moving upstream in the flow; the presence of a chemical signal was necessary.
Although flow cues alone were not sufficient for lobsters to locate an odor
source in our assay, lobsters may use these cues in combination with chemical
cues to orient efficiently to the source of an odorant. Hydrodynamic stimuli
can provide potentially valuable information about the direction and spatial
arrangement of stimuli in the environment, and crustaceans are known to
respond to strong local flows and also to more general cues like wave surge
(Breithaupt et al., 1995;
Nevitt et al., 1995
;
Wilkens et al., 1996
).
However, crustaceans do not rely exclusively on flow cues to locate the source
of an odor stimulus; they also extract important information directly from the
spatial or temporal properties of the chemical signal
(Weissburg and Dusenbery,
2002
). Blue crabs (Callinectes sapidus), for instance,
employ a search strategy that incorporates both chemical and flow cues
(odor-gated rheotaxis) to locate the source of an odor (Weissburg and
Zimmer-Faust, 1993
,
1994
;
Weissburg 2000
;
Webster and Weissburg, 2001
;
Weissburg et al., 2002
;
Weissburg and Dusenbery, 2002
;
Keller et al., 2003
). The
concurrent use of both hydrodynamic and chemical cues results in more
efficient searches with more direct paths and fewer course corrections
(Weissburg and Dusenbery,
2002
; Keller et al.,
2003
). The spiny lobsters in our experiments may have also used
flow cues to orient efficiently to the odor source after the chemical signal
had been detected. However, because this experiment was designed specifically
to examine the chemosensory pathways involved in odor guidance, we cannot
definitively identify the searching strategy employed by the animals in our
assay.
Tactile stimulation resulting from physical contact between the second antennae and the side walls of the flume also did not alter the ability of spiny lobsters to locate the odor source. The overall success rate and search efficiency of animals with immobilized antennae was not different from that of animals with free antennae (Fig. 5). Lobsters with immobilized antennae generally walked straight down the center of the flume without attempting to contact the side wall, suggesting that physical contact with the side wall does not necessarily enhance their success rate or search efficiency.
Why have multiple chemosensory pathways?
The results of our experiments strongly suggest that there is a high degree
of functional overlap between the dual antennular pathways for food
localization behavior. Functional overlap is an important feature of many
sensory systems and can benefit an organism in several important ways
(Derby and Steullet, 2001).
Possession of multiple, overlapping sensors allows an animal to continue to
function normally in the event of loss or damage to a subset of sensors
(Derby and Steullet, 2001
).
Lobsters missing part or the entire aesthetasc region occur in both the field
and laboratory (Harrison et al.,
2001
). Because the acquisition of food is crucial for survival, it
is not surprising that lobsters can use other chemosensory structures besides
the delicate aesthetascs to mediate this important behavior.
A multiplicity of receptors can also extend the range of stimuli that a
lobster is able to detect and increase the sensitivity and resolution of the
system (Derby and Steullet,
2001). Electrophysiological studies have demonstrated that
aesthetasc and non-aesthetasc chemoreceptor neurons respond to the same types
of odorants and have similar response thresholds
(Fuzessery, 1978
;
Thompson and Ache, 1980
;
Cate and Derby, 2002b
). The
combination of inputs from these two pathways may allow for much greater
sensitivity than either pathway alone could provide, as suggested by some of
the results of this study. When tested in the high stimulus concentration,
intact animals took more direct paths to the source than either group of
partially ablated animals (aesthetascs ablated and non-aesthetasc chemo- and
mechanoreceptors ablated), suggesting that the combined input of both
chemosensory pathways provides more information than either pathway alone.
Although each pathway alone is sufficient to drive the behavior in this
instance, the performance of the lobster is enhanced by their combined
activity.
Although functional overlap can have important benefits, it is likely that the aesthetasc and non-aesthetasc chemosensory pathways also have specialized roles that would emerge under different experimental conditions. Despite the lack of experimental demonstrations of specific roles for each pathway in complex behaviors, both the organization of the pathways and the results of behavioral studies with other species of decapod crustaceans provide some possibilities.
The aesthetasc pathway originates in the olfactory receptor neuron
innervating each aesthetasc on the antennule. The axons of these neurons
synapse onto olfactory interneurons within the olfactory lobes of the
deutocerebrum (Schmidt and Ache,
1992,
1996b
;
Sandeman and Mellon, 2002
).
The paired olfactory lobes have a glomerular organization and are structurally
analogous to the olfactory bulbs of vertebrates and the antennal lobes of
insects (Sandeman and Denburg,
1976
; Mellon and Munger,
1990
; Sandeman et al.,
1992
; Schmidt and Ache,
1992
,
1996b
). Glomeruli are typical
features of first-order olfactory processing centers
(Hildebrand, 1995
;
Eisthen, 2002
) and are thought
to play an important role in determining odor quality. Indeed, behavioral
experiments show that the aesthetascs are sufficient to mediate olfactory
discrimination of relevant food odor mixtures
(Steullet et al., 2002
).
Although they are not necessary for analyzing food odors (at least at the
concentrations tested), aesthetascs may be important in determining the
quality of other types of odor stimuli. In the male blue crab, aesthetascs are
essential for mediating the response to courtship and mating signals (Gleeson,
1982
,
1991
). It is possible that the
aesthetasc pathway also functions in spiny lobster intraspecific
communication, perhaps by mediating the response to aggregation signals.
By contrast, the organization of the non-aesthetasc pathway suggests that
it may play a role in detecting spatial aspects of a chemical stimulus. The
non-aesthetasc pathway contains both chemosensory and mechanosensory
afferents, including those from bimodal non-aesthetasc sensilla on the
antennular flagella. Although this pathway is thought to be involved primarily
in driving sensory-motor reflexes and movements of the antennules
(Maynard, 1966;
Schmidt and Ache, 1993
), more
recent work indicates that it also functions in a variety of odor-mediated
behaviors (Steullet et al.,
2001
,
2002
; present study). It has
been hypothesized that the bimodal non-aesthetasc sensilla, which allow spiny
lobsters to detect both chemical and hydrodynamic characteristics of an odor
stimulus, may provide the animal with information about the location of
stimulation on the antennule. Additionally, the stratified organization of the
lateral antennular neuropils (one pair of target neuropils in this pathway)
has been hypothesized to represent a spatial map of sensory inputs on the
antennule (Schmidt and Ache,
1996a
). Although it has not been demonstrated experimentally, the
non-aesthetasc pathway may detect spatial aspects of an odor stimulus through
the integration of chemosensory and mechanosensory cues. The fact that the
output interneurons from the lateral antennular neuropils and from the
olfactory lobes project to distinctly different regions of the protocerebrum
(Sullivan and Beltz, 2001
)
supports the notion that these pathways have some divergent functions.
Possession of multiple chemosensory pathways with redundant as well as complementary functions may allow a lobster to detect and discriminate over a much broader range of chemical stimuli than would be possible with only a single chemosensory pathway. Although unique behavioral roles for either chemosensory pathway in the Caribbean spiny lobster have not yet been conclusively demonstrated, several possibilities for specialized functions exist. Ongoing experiments in our laboratory are focused on these possibilities in order to understand the functional significance of the dual chemosensory pathways of the Caribbean spiny lobster.
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