Non-olfactory chemoreceptors in asymmetric setae activate antennular grooming behavior in the Caribbean spiny lobster Panulirus argus
Department of Biology and Center for Behavioral Neuroscience, Georgia State University, Atlanta, GA 30302-4010, USA
* Author for correspondence (e-mail: Spiny.Lobster{at}gmx.net)
Accepted 25 October 2004
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Summary |
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Key words: chemical senses, antennule, Crustacea, olfaction, spiny lobster, Panulirus argus
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Introduction |
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The most obvious and usually most numerous type of antennular chemosensory
sensilla are the aesthetascs (AE;
Laverack, 1964;
Spencer and Linberg, 1986
;
Grünert and Ache, 1988
;
Cate and Derby, 2001
).
Aesthetascs are long, slender, tube-like setae with an extremely thin cuticle.
They are unique to the antennule, and on the antennule they are restricted to
the lateral flagellum. Aesthetascs are classified as olfactory sensilla, based
on their similarities with olfactory sensilla of insects
(Laverack and Ardill, 1965
;
Spencer and Linberg, 1986
;
Grünert and Ache, 1988
;
Gleeson et al., 1996
). In the
spiny lobster Panulirus argus, aesthetascs occur on each annulus in
the distal third of the lateral flagellum, except for some annuli at the very
tip of the flagellum (Laverack,
1964
; Cate and Derby,
2001
). A mature annulus bears two parallel rows of aesthetascs
with 812 sensilla each. Each aesthetasc is innervated by ca. 300
chemosensory receptor neurons, but no mechanosensory receptor neurons
(Laverack and Ardill, 1965
;
Grünert and Ache, 1988
;
Steullet et al., 2000b
).
Aesthetascs respond to a wide range of chemical stimuli that are typical for
aquatic chemoreceptors (Anderson and Ache,
1985
; Schmiedel-Jakob et al.,
1989
; Michel et al.,
1991
,
1993
;
Steullet et al., 2000b
).
Besides the aesthetascs, numerous other sensilla of different types are
located on the lateral and the medial flagella and the basal segments of the
antennule. Most if not all of these sensillar types represent bimodal, chemo-
and mechanoreceptive sensilla (Laverack,
1964; Derby, 1982
;
Spencer and Linberg, 1986
;
Cate and Derby, 2001
) and are
called `non-olfactory' (Schmidt and Ache,
1996a
) or `non-aesthetasc'
(Steullet et al., 2001
;
Cate and Derby, 2001
)
sensilla. Among the non-olfactory sensilla, two groups can be differentiated:
sensilla that occur on both flagella and on the basal segments, and sensilla
that are restricted to the lateral flagellum and, together with the
aesthetascs, form a conspicuous `tuft' of setae
(Laverack, 1964
;
Spencer and Linberg, 1986
;
Cate and Derby, 2001
). In
spiny lobsters, the first group includes simple smooth setae (of different
lengths), plumose setae, short setuled setae, and hooded setae
(Cate and Derby, 2001
). The
non-olfactory `tuft' sensilla comprise guard setae (GS), companion setae (CS),
and asymmetric setae (AS; Laverack,
1964
; Spencer and Linberg,
1986
; Gleeson et al.,
1993
; Cate and Derby,
2001
). On each mature aesthetasc-bearing annulus reside two guard
setae, 24 companion setae, and one asymmetric seta. The guard setae
flank the distal row of aesthetascs, 1 or 2 companion setae are located
laterally to each guard seta, and the asymmetric seta is located between the
end of the aesthetasc rows and the lateral guard seta
(Laverack, 1964
;
Spencer and Linberg, 1986
;
Gleeson et al., 1993
;
Cate and Derby, 2001
). None of
the tuft setae except for aesthetascs has been analyzed in its ultrastructure
or electrophysiological properties.
Sensory neurons of the aesthetascs and the non-olfactory sensilla project
to two almost entirely separated pathways in the brain. Aesthetascs
selectively innervate the olfactory lobe (OL), a glomerular neuropil of the
deutocerebrum (Sandeman and Denburg,
1976; Mellon and Munger,
1990
; Schmidt and Ache,
1992
; Sandeman and Sandeman,
1994
). The axons of the olfactory lobe projection neurons form a
common fiber tract (olfactory globular tract, OGT) projecting to protocerebral
neuropils in the eyestalk ganglia (Mellon
et al., 1992
; Schmidt and
Ache, 1996b
; Sullivan and Beltz,
2001a
,b
).
These criteria define the aesthetascOLOGT axis as the olfactory
pathway of decapod crustaceans. Chemo- and mechanosensory neurons of the
non-olfactory sensilla on both flagella project to a stratified but
non-glomerular, bilobed neuropil of the deutocerebrum, the lateral antennular
neuropil (LAN) (Schmidt et al.,
1992
; Schmidt and Ache,
1996a
), which also contains the major arborizations of antennular
motoneurons (Maynard, 1965
;
Schmidt and Ache, 1993
;
Roye, 1994
). Thus the
non-olfactory sensilla-LAN pathway represents a second antennular chemosensory
pathway that parallels the olfactory pathway and also functions as the
antennular motor center.
Linking specific, chemically elicited behavioral responses to particular
sensilla types and hence to one of the two pathways has been challenging.
Sensillar ablation experiments in P. argus led to the conclusion that
detection of, orientation to, and associative learning of food-related
chemicals can be mediated by either the olfactory or non-olfactory antennular
pathway (Steullet et al.,
2001,
2002
;
Horner et al., 2004
). The only
specific behavior definitively linked to one of the pathways is elicitation of
courtship behavior in male blue crabs Callinectes sapidus by a female
pheromone (Gleeson, 1982
),
which is elicited only by the aesthetasc pathway.
Recently, another behavior also has been attributed to activation of
aesthetascs: antennular grooming behavior (AGB), produced by many decapod
crustaceans spontaneously without any obvious sensory stimulation and
occurring with an increased frequency after feeding
(Maynard and Dingle, 1963;
Snow, 1973
;
Farmer, 1974
; Bauer,
1977
,
1981
;
Alexander et al., 1980
;
Zimmer-Faust et al., 1984
;
Barbato and Daniel, 1997
;
Daniel et al., 2001
;
Wroblewska et al., 2002
). AGB
is a very distinctive and stereotyped behavior consisting of two major
components. In the first component, called `antennule wiping', both antennular
flagella are repeatedly brought down towards the last pair of mouthpart
appendages, the third maxillipeds. Then they are grabbed by their endopodites
equipped with pad-like structures consisting of densely packed specialized
setae (Farmer, 1974
;
Bauer, 1977
;
Alexander et al., 1980
;
Wroblewska et al., 2002
) and
are repeatedly pulled through these pads. The second component, called `auto
grooming', consists of rubbing movements of the two third maxillipeds against
each other, and usually occurs after a bout of wipes. In several species of
decapod crustaceans, including P. argus, both components of AGB can
be elicited by chemical stimulation, but normally by only one chemical:
L-glutamate (Barbato and Daniel,
1997
; Daniel et al.,
2001
). In a series of sensillar ablation experiments on P.
argus, Wroblewska et al.
(2002
) attempted to identify
the sensilla responsible for the chemical elicitation of AGB. After
establishing that `tuft' sensilla on the lateral flagellum are solely
responsible for the elicitation of AGB by L-glutamate, guard and
companion setae were selectively ablated. From the result of this experiment,
in which the elicitation of AGB by L-glutamate was unaffected, it
was concluded that chemical elicitation of AGB is mediated by aesthetascs.
This conclusion was based on the argument that aesthetascs are far more
numerous than asymmetric setae, the only other sensilla not eliminated by the
ablations (Wroblewska et al.,
2002
).
Here we report further ablation experiments in the spiny lobster Panulirus argus aimed at scrutinizing the conclusion that the aesthetascs, and hence the olfactory pathway, mediate the chemical elicitation of AGB. Selective removal of aesthetascs or asymmetric setae showed that asymmetric setae are necessary and sufficient for the elicitation of AGB and that aesthetascs do not contribute. Based on scanning electron microscopy and confocal microscopy, we provide morphological evidence that asymmetric setae represent typical bimodal chemo- and mechanosensitive sensilla. From these findings, we conclude that AGB is mediated by the non-olfactory sensilla-LAN pathway.
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Materials and methods |
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Chemical stimuli
Stock solutions (0.1 mol l1, pH 7.9) of
L-sodium glutamate were prepared in ASW and stored in aliquot
samples at 20°C until needed. Prior to an experiment, the samples
of stock solution were thawed and diluted to 0.5 mmol l1
with ASW.
All chemicals were obtained from Sigma (St Louis, MO, USA) unless otherwise noted.
Behavioral assays
Animals were tested in individual 16 liter aquaria for elicitation of
antennular grooming behavior (AGB) by application of 3 ml of 0.5 mmol
l1 L-glutamate (in ASW) using a hand-held syringe
with a thin polyethylene tube attached to its tip. The opening of the tube was
placed close to the antennules to minimize dilution of the stimulus. All
experiments were performed at about the same time of the day, in the early
afternoon, with illumination by fluorescent lighting. On each experimental
day, each animal was stimulated 3 times, allowing about 1 h between
stimulations. In the first set of experiments (Exp. 1), we counted the total
number of complete wipes of both antennules for 2 min before introduction of
the stimulus and for another 2 min after stimulation in order to subtract
baseline activity from activity induced by the stimulation. Since the number
of spontaneous wipes in the 2 min before stimulation was extremely small (in a
total of 90 trials on 18 animals, only 1 wipe occurred in the pre-stimulation
period) compared to the induced activity, we only counted the number of wipes
in the 2 min post-stimulation period. The numbers given in the Results section
are based on these counts. A `complete wipe' is defined as a sequence of
movements, in which at least one flagellum of the lowered antennule touched
the third maxillipeds and was actively pulled forward while the third
maxillipeds moved backwards. This excluded occasionally occurring `incomplete
wipes', in which the third maxillipeds executed grasping and backwards pulling
movements typical of wiping, but without actually being in touch with an
antennule. The number of wipes was counted by direct observation during the
experiments. About 10% of the animals that were initially tested for
responsiveness did not respond to the stimulation with L-glutamate
even after some days in the test tanks. These animals were eliminated from the
experiments and replaced by others.
Ablations
To address whether aesthetascs or asymmetric setae are responsible for the
chemical elicitation of AGB, we performed selective bilateral ablations of the
sensilla in question and control experiments, in which other sensilla not
implicated in AGB (the guard setae) were ablated. All ablations were performed
on animals immobilized on a plastic retaining device within a shallow
container of ASW with the proximal region of the lateral flagella stapled to a
Sylgard®-coated platform. Sensilla were removed surgically
under a dissecting scope (SZ40, Olympus: Melville, NY, USA) by cutting them
off at their base using microblades (Fine Science Tools: North Vancouver,
Canada) or with hand-honed minuten pins held by a blade holder. In all cases,
the tip of the lateral flagellum comprising ca. 15 slender annuli was cut off,
since it was not possible to reliably eliminate the sensilla in question from
this region.
Since aesthetascs and asymmetric setae are located within 2 rows of comparatively massive and stout guard setae, it is not possible to surgically remove one of the former sensilla populations without removing at least one row of guard setae. Thus for experimental animals, either the lateral guard setae were removed together with the asymmetric setae (AS-ablation) or the medial guard setae were removed together with the aesthetascs (AE-ablation). To control for a possible effect of the ablation of guard setae and to serve as a general control for possible effects of the surgery itself, either the lateral or medial guard setae were selectively removed in two groups of experimental animals (GS-ablation).
In one experiment, a group of 814 spiny lobsters was tested in the above detailed way for 3 consecutive days. On the following day (or the following 2 days in the case of AE-ablation in Exp. 2, see below), the sensilla in question were surgically removed. In those experiments in which the animals were subdivided into two subgroups that were subjected to removal of different sensilla (Exp. 3 and 4, see below), care was taken to match animals according to their previous responsiveness to avoid a possible bias in one of the treatment groups that might obscure the treatment effect. This `matched selection' was used because of the substantial variation in the baseline responsiveness between individuals. After at least 1 day of rest after surgery, animals were retested for 3 consecutive days. In some experiments, animals were retested in further test periods of 3 consecutive days following the initial retest period over longer time intervals.
In Exp. 1, animals were subjected to two consecutive surgeries. In the first surgery, lateral guard setae were ablated, and after a retest period of 3 consecutive days, asymmetric setae were removed. In Exp. 2, the medial guard setae and the aesthetascs were removed simultaneously. In Exp. 3, two subgroups of animals were treated differently: in one subgroup, lateral guard setae and asymmetric setae were removed simultaneously; in the other subgroup, medial guard setae and aesthetascs were removed simultaneously. In Exp. 4, two subgroups of animals were treated differently: in one subgroup, medial guard setae were removed; in the other subgroup, medial guard setae and aesthetascs were removed simultaneously.
To evaluate the completeness of selective sensilla removal, lateral flagella of all experimental animals were collected and fixed in 4% paraformaldehyde (+ 15% sucrose) for several hours. After rinsing in 0.1 mol l1 Soerensen phosphate buffer (SPB), the flagella were viewed under a high-power dissecting scope (MZ 16, Leica: Wetzlar, Germany), and the number of sensilla that had escaped removal, as well as the number of sensilla that were removed unintentionally (AS, in the case of AE-ablation), were counted. To document the completeness of sensilla removal, lateral flagella were imaged using a high-resolution digital camera (DC 500, Leica: Wetzlar, Germany) attached to the dissecting scope.
Data analysis and statistics
The number of wipes counted in each 2 min post-stimulation period
represents the wipe rate (wipes/2 min). Responses to the three daily
presentations of the standard stimulus were averaged for each individual to
obtain its mean daily wipe rate. The individual mean daily wipe rates were
averaged over the entire population of experimental animals. Differences in
the mean daily wipe rates of the population throughout the duration of the
respective experiment were analyzed using one-way repeated-measures analysis
of variance (RM-ANOVA). Since for the blocks of 3 consecutive days, in which
the test conditions were constant, the mean daily wipe rates of the population
usually did not differ significantly (with only two exceptions: Day 07 and Day
09 in Exp. 1 and Day 08 and Day 10 in AE-ablations of Exp. 3), we averaged the
mean daily wipe rates of the population for each of these blocks. Differences
in the 3-day population means were analyzed using one-way RM-ANOVA and paired
t-tests where appropriate. All statistical tests were performed with
statistics software (GraphPad Prism, GraphPad Software: San Diego, CA,
USA).
Scanning electron microscopy
For scanning electron microscopy, lateral flagella of intermolt specimens
of Panulirus argus were cut off proximal to the tuft region under
Panulirus saline (460 mmol l1 NaCl, 13 mmol
l1 KCl, 13.6 mmol l1 CaCl2, 10
mmol l1 MgCl2, 14 mmol l1
Na2SO4, 3 mmol l1 Hepes, 1.7 mmol
l1 glucose, pH 7.4 adjusted with NaOH, 950 mOsmol) and
cleaned by sonication for ca. 10 min (VWR Model 50T, VWR International: West
Chester, PA, USA). The tuft region was then cut into pieces that were fixed by
immersion in 5% glutaraldehyde (in 0.1 mol l1 SPB + 15%
sucrose) followed by 2% osmium tetroxide (in 0.1 mol l1 SPB)
as described in detail previously (Gnatzy
et al., 1984). After rinsing with SPB for 4x 30 min, the
pieces were dehydrated in an ascending ethanol series (50%, 70%, 85%, 95%,
100%) for 2x 30 min at each concentration. Then the pieces were
incubated for 1 h in hexamethyldisilazane and air dried
(Nation, 1983
). The pieces
were glued to holders, sputtered with palladium (Vacuum Desk II, Denton:
Moorestown, NJ, USA), and viewed in a scanning electron microscope (Stereoscan
420, Leica: Wetzlar, Germany) equipped with digital image capturing (LEO-32,
Leica: Wetzlar, Germany).
Confocal microscopy
For confocal microscopy, lateral flagella of late premolt and early
postmolt specimens of Panulirus argus were cut off proximal to the
tuft region under Panulirus saline and divided into pieces that were
fixed in 4% paraformaldehyde (in 0.1 mol l1 SPB + 15%
sucrose) for at least 4 h. After rinsing with SPB, the pieces were embedded in
gelatin as described in detail previously
(Schmidt et al., 1992). The
gelatin-blocks were hardened with 4% paraformaldehyde overnight at 4°C,
rinsed briefly with SPB and then cut into 50 µm or 70 µm thick sagittal
sections on a vibrating microtome (VT 1000S, Leica: Wetzlar, Germany). The
free-floating sections were incubated overnight in AlexaFluor568-labeled
phalloidin (Molecular Probes: Eugene, OR, USA) at a dilution of 1:200 in SPB
containing 0.3% Triton-X-100. Afterwards the sections were rinsed 3x 30
min in SPB, then incubated for 20 min in Hoechst 33258 diluted 1:100 in SPB
from a stock solution of 1 mg ml1, and after a final rinse
in SPB, coverslips were placed on top of the sections in (1:1) glycerol/SPB
containing 5% diazabicyclol[2.2.2]octane (DABCO) to prevent bleaching. The
sections were viewed and imaged on a confocal microscope (LSM 510, Zeiss:
Jena, Germany) using the associated software package. Stacks of 0.51.0
µm thick optical sections covering the entire section thickness were
collected, and the stacks were then collapsed to produce single
two-dimensional images.
Image processing
All digital images were processed by a graphics program (Paint Shop Pro 5,
Jasc Software: Eden Prairie, MN, USA) before they were arranged to the final
figures using a presentation program (PowerPoint, Microsoft: Redmond, WA,
USA).
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Results |
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In bilateral coordination of wiping as well as the way in which the antennules were grasped by the third maxillipeds, animals showed individually different `styles', to which they adhered over many trials. In the bilateral coordination of wiping, two different patterns could be clearly differentiated: either both antennules were wiped simultaneously, or a bout of wiping of one antennule was followed by a bout of wiping of the other antennule (usually with about the same number of wipes). Alternating wiping of both antennules also occurred but was much rarer. In almost all animals, a typical response contained elements of both major patterns, but the relative proportion of both varied substantially between individuals constituting typical `styles' of wiping patterns. Individual animals differed greatly in the way in which the antennules were grasped: usually both flagella were pulled through the grooming brushes on the third maxillipeds, but sometimes only one was in contact with them (wipes were counted). Rarely (but in some individuals quite frequently) the antennule was not in touch with the maxillipeds but still pulled forward in the typical fashion (wipes not counted); at other times, the antennules were grabbed correctly by the maxillipeds but not pulled while the maxillipeds also failed to execute their normal backward movement (wipes not counted).
As described previously, AGB in the spiny lobster, as in other decapod
crustaceans, is a stereotyped behavior and consists of a sequence of movements
that can readily be distinguished from all other behaviors
(Maynard and Dingle, 1963;
Snow, 1973
;
Farmer, 1974
; Bauer,
1977
,
1981
;
Alexander et al., 1980
;
Zimmer-Faust et al., 1984
;
Barbato and Daniel, 1997
;
Daniel et al., 2001
;
Wroblewska et al., 2002
).
Clearly, the antennules and third maxillipeds are the main appendages involved
in this behavior, and their movements define it. However, according to our
observations, AGB is more complex than that. Frequently, AGB was `assisted' by
coordinated movements of the first 1 or 2 pairs of legs, helping to bring the
antennular flagella down to the maxillipeds. Furthermore, AGB also was usually
accompanied by a change in body posture. In the resting state, the body of the
animals had a horizontal to slightly frontally raised orientation, which upon
stimulation changed to a posture in which the frontal part of the body was
raised considerably to accommodate the backward and downward movement of the
antennules towards the mouthparts. In their resting posture and activity, the
experimental animals also adhered to individually different styles. The
resting posture reached from lying flat on the bottom to standing high on
maximally extended legs, and the resting activity reached from no obvious
locomotor activity (ca. 80% of the animals) to continuous walking and probing
with the legs (ca. 10% of the animals). Both aspects seemed to have a
systematic influence on the magnitude of AGB: animals that had a flat resting
posture showed less intense AGB and much more often failed to respond at all
than animals with a raised resting posture. Animals that were very active
during rest seemed to respond less than more inactive animals with similar
resting posture.
Effect of selective sensilla removal
Experiment 1
Exp. 1 examined the effect of the removal of asymmetric setae (AS) on
chemically elicited AGB (Fig.
1). It started with a period of 3 consecutive days in which the
baseline responsiveness of the animals (N=8) was determined. During
these 3 days, the mean wipe rates increased slightly and continuously, from
11.5±2.3 to 15.1±3.4 wipes/2 min (mean ±
S.E.M.), but these differences were not statistically significant
(P>0.05, RM-ANOVA, NewmanKeuls post-test). Removal
of the lateral guard setae (GS; Fig.
2A), which was required to access the AS for their later removal
but also served as a control for possible non-specific effects of the surgery
itself, did not significantly change the wipe rate on the day following the
surgery (12.2±1.7 wipes/2 min) compared to the last day before surgery.
In the 2 following days, the wipe rate again increased continuously,
surpassing the maximum pre-surgery wipe rate on both days (day 2,
16.1±1.4 wipes/2 min; day 3, 20.7±2.7 wipes/2 min). The
difference in wipe rate between the post-surgery days 1 and 3 was
statistically significant (P<0.01, RM-ANOVA, NewmanKeuls
post-test). Comparing the mean wipe rates between the 3-day pre- and
post-surgery blocks revealed no statistically significant difference
(P>0.05; RM-ANOVA, NewmanKeuls Collectively
post-test). these data demonstrate that (1) AGB is not influenced by
removal of the lateral GS (confirming previous observations by
Wroblewska et al. 2002), (2)
under the experimental conditions used, surgical trauma at most has a small,
non-significant negative effect on AGB, and (3) independent of sensilla
removal, the mean wipe rate tends to increase over the 3 consecutive days of
an experimental block.
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The subsequent removal of the AS (Fig. 2B) caused a dramatic and statistically significant reduction of the mean wipe rate. From 1.0±0.6 wipes/2 min on post-surgery day 1, the wipe rate dropped further and actually reached 0 on post-surgery day 3. Comparing the mean wipe rates between the 3 day pre- and post-AS-ablation blocks showed that this difference is significant statistically (P<0.001, RM-ANOVA, NewmanKeuls post-test). This result demonstrates that AS are necessary for the chemical elicitation of AGB.
Retesting ablated animals after longer time intervals (ca. 3 weeks, Days 3537; 5 weeks, Days 4951; 6 weeks, Days 5658, after the post-AS-ablation block; none of the animals molted during this time) showed a small and sustained increase in the mean wipe rates with respect to the 3-day post-AS-ablation period; nonetheless, wipe rates remained substantially lower than in the pre-AS-ablation period (maximum mean wipe rate as measured on the last test day: 4.3±0.8 wipes/2 min). Comparing the mean wipe rates between the 3-day test blocks revealed that at all three longer time intervals, the mean wipe rate was significantly different from the mean wipe rate of the 3-day pre-AS-ablation block (P<0.001 for all three time intervals; RM-ANOVA, NewmanKeuls post-tests) but also from the mean wipe rate of the 3-day post-AS-ablation block (P<0.05 for all three time intervals; RM-ANOVA, NewmanKeuls post-tests). These results suggest that chemically elicited AGB functionally recovers to some extent after longer time intervals even without any possible regeneration of the ablated AS. Since the wipe rates in this time do not increase systematically but stay at a rather constant and substantially lower level than before removal of the AS, this functional recovery represents a partial one at best.
Counting the number of AS that had escaped selective removal after the end of the experiments, showed a shaving efficiency of about 97% (Table 1, Fig. 2B).
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Experiment 2
Exp. 2 examined the effect of the removal of aesthetascs (AE) on chemically
elicited AGB (Fig. 3). It also
started with a period of 3 consecutive days in which baseline responsiveness
of animals (N=8) was determined. In contrast to Exp. 1, the mean wipe
rates stayed rather constant during this time period. Since Exp. 1 had shown
that removal of one row of GS has no significant effect on the elicitation of
AGB, we selectively removed the medial row of GS guard setae together with the
aesthetascs (AE) in a single procedure
(Fig. 2C). Retesting animals on
3 consecutive days after this surgery showed responses that were very similar
to those obtained during the pre-AE-ablation block. Comparing the mean wipe
rates between the 3-day pre- and post-AE-ablation blocks revealed no
statistically significant difference (P>0.05; RM-ANOVA,
NewmanKeuls post-test). Within the post-AE-ablation block, the
mean wipe rate showed a small, statistically non-significant increase. This
result demonstrates that AE are not necessary for the chemical elicitation of
AGB.
|
Retesting ablated animals after longer time intervals (1 week, Days 1517; 2 weeks, Days 2224, after the post-AS-ablation block; none of the animals molted during this time) showed a substantial and sustained increase in the mean wipe rates with respect to the 3-day post-AE-ablation period. Comparing the mean wipe rates between the 3-day test blocks revealed that at both longer time intervals, the mean wipe rate was significantly different from the mean wipe rate of the 3-day post-AE-ablation block (1 week, P<0.01; 2 weeks, P<0.05; RM-ANOVA, NewmanKeuls post-test), whereas it did not differ significantly between the two longer time intervals (P>0.05; RM-ANOVA, NewmanKeuls post-test). This unexpected result suggests that removal of the AE might have a long-term positive effect on chemically elicited AGB. Since no proper controls (sham-ablated or GS-ablated animals) were run in parallel in this experiment, we did an additional experiment (Exp. 4, see below) to specifically address this question.
Counting the number of AE that had escaped selective removal after the end of the experiments, showed a shaving efficiency of about 99.9% (Table 1, Fig. 2C). Since shaving the AE carries a relatively high risk of accidentally removing some AS, we also counted the number of missing AS and found that on average the experimental animals had lost about 29% of the entire AS population in the shaving (Table 1, Fig. 2C).
Experiment 3
Exp. 3 addressed the concern that the different effects of selective AS and
AE removal on chemically elicited AGB as seen in Exp. 1 and 2, could be
partially due to uncontrolled factors since these experiments were performed
sequentially on two different groups of animals. Therefore in Exp. 3, we did
the selective ablations in parallel on different members of a third group of
animals. To avoid any bias due to the substantial inter-individual differences
in responsiveness (see above), we did not assign the animals (N=12)
to one of the two treatments before the experiment but based this decision on
their performance during the initial period of 3 consecutive days, in which
the baseline responsiveness was determined. After this 3-day block, we paired
similarly responsive animals and subjected one arbitrarily chosen member of
each pair to the selective removal of AS (and lateral GS) and the other member
to the selective removal of AE (and medial GS).
Exp. 3 started with a period of 3 consecutive days in which the baseline responsiveness of the animals was determined (Fig. 4). As in Exp. 1, the mean wipe rates increased continuously slightly but non-significantly during these 3 days in both groups of animals, from 13.6±4.3 to 21.1±4.1 wipes/2 min in the animals that got their AS shaved afterwards, and from 14.2±2.1 to 18.9±4.9 wipes/2 min in the animals that got their AE shaved afterwards.
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Subsequent removal of AS (together with lateral GS) in one group of animals (N=6) and of AE (together with medial GS) in the other group of animals (N=6) had drastically different effects on chemically elicited AGB. In animals without AS, the mean wipe rate dropped substantially to reach 0 on post-surgery day 2, with a small rebound on post-surgery day 3 where the mean wipe rate increased to 1.9±0.6 wipes/2 min. Comparing the mean wipe rates between the 3-day pre- and post-AS-ablation blocks showed that this difference is statistically significant (P<0.01, paired t-test). In contrast, the mean wipe in the animals without AE dropped only slightly on the first post-surgery day (the difference between this day and the last day before the surgery was not statistically significant) and then rebounded to reach a value on post-AE-ablation day 3, slightly but not significantly higher than on the last day before the surgery. Comparing the mean wipe rates between the 3-day pre- and post-AE-ablation blocks showed no statistically significant difference between them (P=0.82, paired t-test). Comparing the post-ablation mean wipe rates between the two groups of animals revealed a significant difference between them (P<0.0001, unpaired t-test). In summary, the results obtained in Exp. 3 fully support the main results from Exp. 1 and 2: selective removal of AS causes a dramatic decrease in chemically elicited AGB (to almost zero), whereas selective removal of AE has no detectable effect on AGB. This supports the conclusion that AS are necessary and sufficient for driving chemically elicited AGB and that AE have no role in eliciting this behavior.
Counting the number of AS and AE, respectively, that had escaped selective removal after the end of the experiments revealed shaving efficiencies of about 99% for the AS-ablated animals and 99.9% for the AE-ablated animals (Table 1).Counting the number of AS that had been removed accidentally in the AE-ablated animals revealed that on average they had lost about 20% of the entire AS population in the shaving (Table 1).
Experiment 4
Exp. 4 examined the possibility that removal of AE has a long-term positive
effect on chemically elicited AGB as indicated by the results of Exp. 2. To
test this possibility, we set up a new group of 14 spiny lobsters, half of
which were subjected to the same treatment as in Exp. 2 in that their AE were
specifically removed (together with the medial GS) whereas the other half
served as a control and received a selective shaving of only the medial GS. To
avoid any bias due to the substantial inter-individual differences in
responsiveness, we assigned the animals to one of these treatments based on
their baseline responsiveness in the same way as described for Exp. 3.
Exp. 4 started with a period of 3 consecutive days in which the baseline responsiveness of the animals was determined (Fig. 5). As in Exp. 1 and 3, the mean wipe rates increased slightly but non-significantly during these 3 days in both groups of animals from 12.1±2.6 to 19.2±3.8 wipes/2 min in the animals that got their AE shaved afterwards, and from 14.1±2.6 to 20.8±3.7 wipes/2 min in the animals that got their medial GS shaved afterwards.
|
The subsequent removal of AE (together with medial GS) in one group of animals (N=7) and of only medial GS in the other group of animals (N=7) had no effect on chemically elicited AGB, including retests of the ablated animals after longer time intervals (3 days: Days 1315, and 10 days: Days 2022, after the post-ablation block). This observation is supported by statistical analyses. Comparing the mean daily wipe rates or the mean wipe rates averaged over the 3-day blocks revealed no statistically significant differences for either group of animals (P=0.36 for AE-ablated animals; P=0.80 for GS-ablated animals; RM-ANOVA). There also was no statistically significant difference between the mean post-ablation wipe rates (averaged over the 3-day blocks) of both groups of animals (P=0.81, Days 0810; P=0.73, Days 1315; P=0.88, Days 2022; unpaired t-tests). From this result, we conclude that removal of AE has no effect, also in particular not any positive effect, on chemically elicited AGB, as was suggested by the result of Exp. 2. This in turn indicates that the increased mean wipe rate observed in Exp. 2 at long time intervals after removal of the AE is most likely the result of an uncontrolled factor (see Discussion for a possible explanation).
Counting the number of AE that had escaped selective removal after the end of the experiments showed a shaving efficiency of about 99.9% (Table 1). Counting the number of AS that had been removed accidentally in AE-ablated animals showed that on average they had lost about 15% of the entire AS population in the shaving (Table 1). In the GS-ablated animals, AS were missing on some annuli (maximally 6), supposedly due to natural wear and tear, since the lateral side of the flagella was not touched in the shaving.
Morphological features of asymmetric setae relevant for sensory transduction
Our behavioral experiments lead to the conclusion that in P.
argus, the asymmetric setae are solely responsible for the elicitation of
AGB by chemical stimulation. To understand the sensory basis of AGB in more
depth, we analyzed the morphology of asymmetric setae to search for
modality-specific features, which have been described for diverse sensilla
types of decapod crustaceans (Schmidt and
Gnatzy, 1984). Confocal fluorescence microscopy, which revealed
the cuticular apparatus of the asymmetric setae due to its wide-spectrum
autofluorescence (excitation wavelength 450 nm; recorded emission >500 nm),
in conjunction with bright field light microscopy and scanning electron
microscopy, showed that the asymmetric setae have a smooth, slender shaft
tapering only very gradually towards the tip (Figs
6C,D,F,
7A,B,D). The setal shaft is
about 400 µm long (384±23 µm, mean ± S.D.,
N=8) and its maximum diameter at the base is about 15 µm
(15.5±2.2 µm, mean ± S.D., N=8). A
morphological feature distinguishing the setal shaft of AS from other sensilla
with smooth shafts (GS, CS) is that it is not straight or gradually curved but
has two noticeable kinks, one at about one third of the total length (Figs
6C,D,
7A) and one close to the tip,
at about 95% of the total length (Figs
6F,
7A). The angle of the first
kink is about 13°; the angle of the second kink is about 30°. Distal
to the second kink the shaft changes its shape from cylindrical to laterally
flattened. Both kinks are in roughly the same plane and cause the shaft to
project mesially in between the two rows of aesthetascs. The setal base of AS
is located within a socket structure that lies in a slight depression of the
cuticle (Figs 6D,
7B,E). The cuticular depression
forms a tight bulge around the lateral and distal aspects of setal base, but
is flatter and thus leaves a larger gap at its proximal aspect. The setal
shaft inserts in the bulge with an angle of approximately 70° pointing
distally. A central canal passes through the shaft on its entire length ending
in a terminal pore (Fig. 6E,F).
The terminal pore has a diameter of ca. 0.1 µm and is located at the bottom
of a cuticular ring with a diameter of ca. 1 µm. A terminal pore has been
identified as a modality specific structure of bimodal chemo- and
mechanosensory sensilla in decapod crustaceans, and it is thought that the
pore allows chemical stimuli to access the tips of the dendrites ending below
it (Schmidt and Gnatzy,
1984
).
|
|
Labeling vibrating-microtome sections cut sagittally through the lateral
flagellum with the f-actin marker phalloidinAlexaFluor568 revealed a
short, phalloidin-positive tube-like structure associated with the base of AS
(Fig. 7) and other
non-olfactory sensilla types of the tuft region (GS, CS) but lacking at the
base of aesthetascs. Since in insect sensilla phalloidin strongly labels the
scolopale (Wolfrum, 1990), a
tube-like supporting structure within the innermost sheath cell surrounding
the transitional region of the dendrites, and because the phalloidin-positive
structures associated with the base of AS, GS and CS have the size and
position known for the scolopales of other sensilla of decapod crustaceans
(Schmidt and Gnatzy, 1984
), we
conclude that the phalloidin-positive structures identified here represent
scolopales. The scolopale of AS is 3040 µm long and has a maximum
diameter of 1015 µm. It consists of relatively fine longitudinal
strands of phalloidin-positive material that only in the middle region form an
almost closed tube-like structure (Fig.
7C,F,H). Analyzing animals in different molt stages (from late
premolt to early postmolt; intermolt animals could not be analyzed since the
hardened cuticle cannot be cut with a vibrating microtome) revealed that the
position of the scolopale in the setal base region changes systematically with
the molt stage. In late premolt animals, in which a new cuticle is already
formed below the old one (N=2), the scolopale was located in the base
of the shaft above the socket (Fig.
7G,H). In very early postmolt animals (1 day after molting,
N=2), the scolopale was also located in the base of the shaft, but
sometimes closer to the socket than in the late premolt animals
(Fig. 7E,F). In animals that
were about 1 week postmolt (N=2), the scolopale was consistently
located below the socket (Fig.
7BD). A scolopale has been identified as a modality
specific structure of mechanosensory and of bimodal chemo- and mechanosensory
sensilla in decapod crustaceans (Schmidt
and Gnatzy, 1984
).
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Discussion |
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Collectively, the results of Exp. 1, 2 and 3 clearly show that chemically
elicited AGB is driven exclusively by the asymmetric setae and that
aesthetascs do not contribute to the elicitation of this behavior. Selective
elimination of the AS as achieved in Exp. 1 and 3 with a completeness of about
97% and 99%, respectively, while leaving the vast majority of aesthetascs
intact, almost completely eliminated chemically elicited AGB. This
demonstrates that the asymmetric setae are necessary for driving AGB.
Conversely, selective elimination of the aesthetascs, as was achieved in Exp.
2, 3 and 4 with 99.9% efficiency while leaving the majority of AS (ca. 70%,
80%, 85%, respectively) intact, had no detectable effect on chemically
elicited AGB (at least during the first 3-day post-ablation block). This
demonstrates that, contrary to the previous conclusion
(Wroblewska et al., 2002), the
aesthetascs are not necessary for driving chemically elicited AGB. Since in
the extensive ablation experiments performed by Wroblewska et al.
(2002
) and in the experiments
presented here (selective ablation of guard setae) no other sensilla types
have been found that are required for elicitation of AGB, it follows that the
AS are not only necessary but also sufficient for driving AGB.
The results of the long-term observations in Exp. 2 indicated a positive influence of selective asymmetric setae removal on AGB, which might be interpreted as an inhibition of AGB mediated by aesthetasc input. Testing this possibility directly in Exp. 4, however, showed no effect of selective aesthetasc removal compared to the previous responses of these animals or compared to the responses of control animals in which only the medial guard setae had been removed. From this finding, we conclude that the aesthetascs are also not mediating any kind of inhibition of AGB and thus seem not to be involved in the elicitation of AGB at all. This poses the question of how the long-term increase in wipe-rate after selective aesthetasc removal in Exp. 2 can be explained. Comparing the responses obtained in all four experiments it becomes obvious that the responsiveness in the initial 3-day test period remained almost constant in Exp. 2, whereas it systematically increased in the other experiments, resulting in an overall reduced level of responsiveness in the initial 3-day test period of Exp. 2 compared to the others. The animals in Exp. 2 had the shortest acclimatization period in the test tanks before the start of the experiments, and we assume that their unusually low and constant responsiveness during the initial test period could result from this. Then the considerably higher (almost doubled) responsiveness after longer time intervals might merely represent a delayed full acclimatization to the test situation. This interpretation is strengthened by the fact that already during the first post-ablation test period the responsiveness rises systematically as it did in the pre-ablation test period in the other experiments. Thus the long-term increase in wipe-rate after selective aesthetasc removal in Exp. 2 likely represents an experimental artifact.
Another unexpected observation was that weeks after an initial almost total
loss of responsiveness following the selective removal of asymmetric setae,
chemical elicitation of AGB showed a partial functional recovery. To attribute
this functional recovery to a possible regeneration of the ablated AS seems
highly unlikely since sensilla formation in decapod crustaceans is only
possible through molting (e.g. Sandeman
and Sandeman, 1996; Steullet
et al., 2000a
), which did not occur in the test animals during the
experiment. Thus, two other explanations are more probable. The first is that
very few remaining AS (ca. 2 or 1 per lateral flagellum in Exp. 1 and 3,
respectively) could strengthen their connections in the CNS and thereby
achieve a much higher effectiveness in eliciting AGB than they had prior to
and immediately after selective AS removal. The second is that other sensillar
types on the lateral flagellum, which project to the same neuropil as AS (most
likely the LAN, see below) but normally do not connect to the interneurons
and/or motoneurons that receive AS input and mediate AGB, start to establish
such connections when the sensory axons of the AS fall silent or begin to
degenerate. We assume that the second possibility is the more likely one,
since the partial recovery of the chemical elicitation of AGB occurred in all
experimental animals, including those in which re-checking confirmed that no
AS were left on both flagella (two animals). Since responses to
L-glutamate are quite common in extracellular recordings from
sensory axons in the lateral flagellum of P. argus
(Laverack, 1964
;
Johnson and Ache, 1978
;
Derby et al., 1991
), it is
conceivable that not only the AS (see below) but also other non-olfactory
sensillar types respond to L-glutamate.
To our knowledge, the findings reported here represent the second case in
which in decapod crustaceans a chemically mediated behavior can be attributed
to only one identified type of sensilla. The other case is the courtship
behavior of male blue crabs Callinectes sapidus, elicited by a sex
pheromone released by pre-molt females and driven by aesthetascs but not by
asymmetric setae (Gleeson,
1982). Several studies on decapod crustaceans have aimed to
identify sensillar types driving specific chemically mediated behaviors
(Steullet et al., 2001
,
2002
;
Wroblewska et al., 2002
;
Keller et al., 2003
;
Horner et al., 2004
). In all
of these studies (except Wroblewska et
al., 2002
), it was found that several sensillar types, including
those providing input to different sensory pathways in the brain (see below),
contribute to the analyzed behaviors, food search, orientation and associative
learning. This large degree of redundancy and functional overlap of sensilla
contributions may be due to the complexity of the observed behaviors and/or
the complexity of the stimulants eliciting them. In contrast, AGB and the
courtship dance of Callinectes sapidus are more stereotyped behaviors
elicited by one key compound (L-glutamate and female sex pheromone,
respectively) making it more conceivable that one sensillum type suffices to
drive them. This conforms to one principle of chemosensory coding known as
`labeled line' (e.g. Ache,
1991
): one key compound leads to the activation of specific
sensory neurons, which causes the activation of specific interneurons in the
CNS, which in turn leads to the elicitation of a specific, often stereotyped
behavior.
While it is surprising that the most numerous sensilla on the lateral
flagellum, the aesthetascs, have been shown only once
(Gleeson, 1982) to be
necessary for driving specific behaviors, it may be equally unexpected to find
the least numerous sensilla, the asymmetric setae (ca. 80 per flagellum in
P. argus), to be not only necessary but also sufficient for driving a
prominent and specific behavior, AGB. Due to their exposed position, the
lateral flagella are often damaged distally and also lose sensilla, including
AS, more proximally by wear and tear
(Harrison et al., 2001
; this
study), raising the question of how reliable the elicitation of AGB by AS is,
or how many AS are sufficient to drive it. Although we did not study this
question directly, the accidental removal of some AS in the specific shaving
of the aesthetascs (Exp. 2, 3 and 4) is informative in this context. In Exp. 2
the highest percentage of accidentally removed AS occurred (on average ca. 30%
of the entire population), but this had no negative effect on the wipe-rate
observed after the sensilla ablation indicating that the remaining 70% of AS
are sufficient to drive AGB. On the other extreme, the maximal average number
of AS escaping selective removal was 3.3% (Exp. 1), and as the result of Exp.
1 clearly demonstrates this number was not sufficient to drive AGB. Thus we
conclude that the minimal number of AS sufficient for driving AGB must lie
between ca. 3 and 70% of the entire population.
Sensory pathway mediating AGB
The identification of the asymmetric setae as driving AGB, leads to a
revision of the hypothesis developed by Wroblewska et al.
(2002) that AGB is mediated by
the central olfactory pathway. It instead strongly suggests that AGB is
mediated by the second antennular sensory pathway in the deutocerebrum, which
parallels the olfactory pathway and is comprised of the lateral and medial
antennular neuropils (LAN, MAN; Maynard,
1965
; Schmidt et al.,
1992
; Schmidt and Ache, 1996). Several lines of evidence support
this new interpretation, which was originally proposed by Barbato and Daniel
(1997
).
(1) The available evidence from labeling sensilla with radioactive leucine
and backfilling axon bundles in the antennular nerve with neuronal tracers
strongly suggests that the axons of the aesthetascs specifically and
exclusively target the ipsilateral olfactory lobe, whereas the afferent axons
from the non-olfactory sensilla on the antennular flagella project to the
ipsilateral LAN (Sandeman and Denburg,
1976; Mellon and Munger,
1990
; Schmidt and Ache,
1992
; Schmidt et al.,
1992
; Sandeman and Sandeman,
1994
). As there is no reason to suggest that the afferents of the
AS deviate from this scheme, we conclude that most likely they also project to
the LAN.
(2) The LAN and MAN contain the major arborizations of the antennular
motoneurons, whereas the olfactory lobe is completely devoid of them
(Maynard, 1965;
Schmidt and Ache, 1993
;
Roye, 1994
). This means that
LAN and MAN represent the motor center for the antennules, the main appendages
executing AGB. Therefore the presumptive afferent projections of the AS to the
LAN, together with the close spatial juxtaposition of sensory afferents and
motoneuron arborizations in the LAN
(Schmidt et al., 1992
;
Schmidt and Ache, 1993
), allow
that a direct sensory-motor coupling underlies the activation of antennular
wiping movements by the chemical stimulation of the AS. This would represent a
far simpler pathway than that proposed by Wroblewska et al.
(2002
), involving the
olfactory lobe activated by aesthetasc input, higher brain centers in the
lateral protocerebrum innervated and activated by ascending OL projection
neurons (Mellon et al., 1992
;
Schmidt and Ache, 1996b
;
Sullivan and Beltz,
2001a
,b
),
and finally the antennular motoneurons residing in the LAN/MAN activated by
descending projection neurons from these protocerebral neuropils.
(3) AGB, although representing a highly stereotyped behavior, cannot be
considered as a simple reflexive behavior since it involves the coordinated
movements of not only the antennules but also other appendages like the third
maxillipeds and the anterior walking legs. To achieve this coordination, the
LAN/MAN neuropils have to be neuronally connected with the motor centers
controlling the movements of the third maxillipeds and the walking legs, which
reside in the suboesophageal ganglion and the thoracic ganglia, respectively
(e.g. Wiens, 1976). Descending
as well as ascending projection neurons, with axons in the circumoesophageal
connectives and arborizations in LAN/MAN that could provide the neuronal
substrate for the observed coordination, have been identified in P.
argus (Schmidt and Ache,
1996a
) and other decapod crustaceans
(Glantz et al., 1981
;
Arbas et al., 1988
), whereas
such neurons are very rare for the olfactory lobe
(Schmidt and Ache, 1996b
).
(4) In addition to the coordination with the third maxillipeds and the
anterior walking legs, both antennules also show coordination with one another
in AGB, which is most obvious when both antennules are wiped simultaneously.
The most likely explanation for this bilateral coordination is a direct
neuronal connection between both sides of the brain. Such connections are
indeed numerous between the LAN/MAN of both sides, since several antennular
motoneurons have minor arborizations on the contralateral side (with respect
to their axon and main arborizations), and many descending and ascending
projection neurons have bilateral arborizations in the LAN/MAN
(Hamilton and Ache, 1983;
Arbas et al., 1988
;
Schmidt and Ache, 1993
;
Schmidt and Ache, 1996a
). By
contrast, only a few neurons have been found that directly connect the
olfactory lobes of both sides of the brain
(Schmidt and Ache, 1996b
).
Functional morphology of asymmetric setae
Our analysis of the morphological features of the asymmetric setae led to
the identification of two modality specific structures, a terminal pore at the
tip of the setal shaft and a scolopale below its base. With these two
prominent features, the AS can be classified as bimodal chemo- and
mechanosensory sensilla typical of decapod crustaceans
(Schmidt and Gnatzy, 1984).
The terminal pore presumably provides access for chemical stimuli to the tips
of the dendrites located below, and the scolopale is interpreted as a rigid
structural element that is involved in mechanosensory transduction likely
taking place in the transitional region of the dendrites
(Moran et al., 1977
;
Crouau, 1982
;
Eberl et al., 2000
).
Since a terminal pore is indicative of chemosensory function, its presence
in the AS corroborates the conclusion from the ablation experiments that the
chemical stimulus eliciting AGB is detected by these sensilla.
L-Glutamate is by far the most potent substance eliciting AGB
(Barbato and Daniel, 1997). AS
are therefore expected to contain chemosensory neurons responding to
L-glutamate. While it is known that the lateral flagellum of P.
argus contains numerous L-glutamate-sensitive units
(Laverack, 1964
;
Johnson and Ache, 1978
;
Derby et al., 1991
) and with
biochemical assays specific, independent binding sites for
L-glutamate have been identified
(Burgess and Derby, 1997
), it
will require selective recordings from AS to characterize the glutamate
sensitivity of their chemosensory neurons, including why such a high
concentration of L-glutamate (>0.1 mmol l1) is
necessary for eliciting AGB (Barbato and
Daniel, 1997
).
The presence of a scolopale in the base region of the asymmetric setae
indicates an additional mechanosensory function. The general notion is that
simple setae of decapod crustaceans do not respond to subtle stimuli such as
water movements, but are rather insensitive and require direct touch to be
stimulated (Garm et al.,
2004). The very tight articulation of the setal shaft of the AS in
the socket and the surrounding cuticular bulge is consistent with this scheme
and suggests that they need direct touch for activation. Given that the AS are
located within the rows of very stout guard setae, they are well shielded from
touching larger outside objects when the antennule moves, suggesting that they
are activated in other ways. The execution of AGB appears to be one likely way
in which the AS could be activated since the antennular flagella are
forcefully pulled through the third maxillipeds during this behavior, which
likely causes a deflection of the AS. Another possible mode of direct
mechanical stimulation of the AS is suggested by the direction in which their
setal shaft faces. The shaft of the AS is articulated and kinked in such a way
as to project `inwards' towards the rows of aesthetascs and to come into
direct contact with about half of the aesthetascs in one row. This
construction indicates that the AS are loosely mechanically coupled to the
shafts of the aesthetascs and that they could be activated when several
aesthetascs move simultaneously. Such simultaneous movements of the
aesthetascs occur regularly not only in AGB but also in antennular flicking
(Gleeson et al., 1993
;
Koehl et al., 2001
). Thus the
AS seem to be well suited to monitor the displacement of the aesthetascs
during flicking and possibly other antennular movements, a function that
appears to be quite useful given that the aesthetascs themselves lack
mechanosensory innervation (Grünert
and Ache, 1988
). It is conceivable that in this indirect way the
AS might also be able to detect `abnormal' movement patterns of the
aesthetascs caused, for instance, by epibiotic organisms growing on them or by
larger objects lodging between them. If this is so, then AS could elicit AGB
not only upon chemical but also upon this kind of mechanical stimulation. The
possibility of a mechanical elicitation of AGB has not been studied in detail
so far, but has been reported to occur in crabs
(Snow, 1973
).
Changes in the position of the scolopale related to the molt stage, as we
have found for the asymmetric setae, have not been previously reported in
decapod crustaceans. The position of the scolopale within the cuticular
apparatus of a sensillum can provide clues as to which mechanical parameter is
the adequate stimulus. In sensilla equipped with a scolopale, sensory
transduction probably occurs in the transition region of the dendrites
surrounded by it (Moran et al.,
1977; Crouau,
1982
; Eberl et al.,
2000
). Thus, the transformation of the outside mechanical stimulus
into a mechanical deformation of the dendrites can only occur distal to the
scolopale. In all cuticular sensilla of decapod (and other) crustaceans in
which a scolopale has been identified, it is always located below the socket
(e.g. Schöne and Steinbrecht,
1968
; Ball and Cowan,
1977
; Altner et al.,
1983
; Espeel,
1985
; Cate and Roye,
1997
), allowing that displacement of the shaft within the socket
acts as the adequate stimulus. Consequently this mode of stimulus
transformation is believed to represent the general principle in
mechanosensitive setae of crustaceans
(Crouau, 1982
), as well as
other arthropods (Barth and Dechant,
2003
). In contrast, a position of the scolopale within the shaft,
which has never been reported before in sensilla of decapod crustaceans, would
not be consistent with this model; it rather suggests that bending of the
shaft serves as an adequate stimulus, as has been speculated previously
(Crouau, 1982
). We interpret
the finding that, several days after molting the scolopale of the asymmetric
setae is located proximal to the socket region, as reflecting the intermolt,
physiologically mature situation. Thus in the functional state of the AS,
displacement of the shaft within the socket could serve as adequate stimulus.
The position of the scolopale in the basal region of the shaft, on the other
hand, seems to be solely a molt-related phenomenon, since we observed this
position only in late premolt and very early postmolt animals. In premolt
animals, the dendrites of sensory neurons pass through the newly formed
cuticular apparatus to the old one, which continues to represent the
functional sensillum until ecdysis occurs (e.g.
Kouyama and Shimozawa, 1984
;
Espeel, 1986
). Therefore a
scolopale position within the newly formed shaft is still proximal to the old,
mechanically active cuticular apparatus and would continue to allow that
displacement of the shaft within the socket serves as adequate stimulus. Only
during the first days after ecdysis, when the scolopale remains within the
base of the shaft, would displacement of the shaft within the socket not be an
adequate stimulus.
Function of antennular grooming
Antennular grooming is widespread among marine decapod crustaceans and
often, like in P. argus, the antennules are the most frequently
groomed appendages or body parts, suggesting that AGB plays a prominent role
in maintaining the function of the aesthetascs
(Maynard and Dingle, 1963;
Snow, 1973
;
Farmer, 1974
; Bauer,
1977
,
1981
;
Alexander et al., 1980
;
Zimmer-Faust et al., 1984
;
Barbato and Daniel, 1997
;
Daniel et al., 2001
). Two
likely complementary functions of AGB have been proposed, both based on the
idea that pulling the flagella through the grooming pads of the third
maxillipeds leads to a mechanical combing of the aesthetascs. One proposed
function is the removal of food particles trapped between aesthetascs during
feeding (Barbato and Daniel,
1997
; Daniel et al.,
2001
). This interpretation is supported by the observation that in
many decapod crustaceans, including P. argus, long bouts of AGB occur
after feeding, whereas at other times the frequency of spontaneous AGB is
rather low (Maynard and Dingle,
1963
; Snow, 1973
;
Zimmer-Faust et al., 1984
;
Barbato and Daniel, 1997
;
Daniel et al., 2001
). It also
is consistent with the robust and selective elicitation of AGB by chemical
stimulation (Zimmer-Faust et al.,
1984
; Barbato and Daniel,
1997
; Daniel et al.,
2001
; Wroblewska et al.,
2002
; this study). The second proposed function of the mechanical
combing caused by AGB is the removal of epibiotic organisms from the
aesthetascs, which otherwise would cause fouling. Supporting evidence for this
interpretation is the observation that epibiotic organisms indeed occur on the
aesthetascs (Shelton, 1974
;
Bauer, 1977
,
1978
) and that their number
increases dramatically when AGB is prevented by ablation of the third
maxillipeds (Bauer, 1977
,
1978
; P. Daniel, personal
communication). In one experiment on shrimps, in which AGB was prevented for
several weeks, all aesthetascs were destroyed; however no explanation as to
how epibiotic fouling could have caused this dramatic effect was provided
(Bauer, 1977
). We propose
another possible function of AGB in the maintenance of aesthetascs. In diverse
decapod crustaceans, including spiny lobsters, large openings of exocrine
epithelial glands have been identified at the base of the guard setae
(Derby, 1982
;
Gnatzy, 1984
;
Spencer and Linberg, 1986
).
Furthermore, smaller pores, which likely represent gland openings as well,
frequently occur at the base of the aesthetascs
(Derby, 1982
;
Fontaine et al., 1982
;
Gleeson, 1982
), suggesting
that epithelial glands of a different type are additionally present in the
tuft region of the lateral flagellum. We hypothesize that the excretions
produced by these `tuft glands' provide chemical antifouling agents and/or
substances that aid in the stabilization/protection of the extremely delicate
cuticle of the aesthetascs. We further hypothesize that AGB not only causes
the mechanical removal of food particles and/or epibiotic organisms but also
provides the means by which the excretions of the `tuft glands' are
distributed over the entire length of the aesthetascs. This hypothesis would
more easily explain why prevention of AGB caused the total loss of aesthetascs
in a relatively short period of time in the experiments on shrimps
(Bauer, 1977
), and it is
supported by the observation that in P. argus a layer of
electron-dense material of hitherto unknown origin covers the aesthetasc
cuticle (Grünert and Ache,
1988
).
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Acknowledgments |
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References |
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