Odor-modulated orientation in walking male cockroaches Periplaneta americana, and the effects of odor plumes of different structure
Department of Biology, Case Western Reserve University, Cleveland, OH 44106-7080, USA
* Author for correspondence (e-mail: maw27{at}po.cwru.edu)
Accepted 29 November 2004
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Summary |
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Key words: olfaction, orientation, cockroach, Periplaneta americana, (-)-periplanone-B, pheromone
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Introduction |
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Insects tracking plumes of airborne odors in flight
(Arbas et al., 1993;
Willis and Arbas, 1997
) or
while walking (Kennedy and Moorehouse,
1969
; Tobin and Bell,
1986
; Bell and Kramer,
1980
; Rust and Bell,
1976
; Wolf and Wehner,
2000
) orient their movement into the wind, while in the absence of
odor they typically show no preferred orientation direction or orient their
movement with the wind. Similar behavior has been observed in swimming fish
(Baker et al., 2002
;
Johnsen and Teeter, 1985
),
walking crustaceans (Grasso and Basil,
2002
; Weissburg and
Zimmer-Faust, 1994
), crawling sea stars
(Moore and Lepper, 1997
) and
crawling snails (Susswein et al.,
1982
; Brown and Rittschoff,
1984
) orienting to odors in flow in marine environments. Thus, an
orientation to environmental flow that is modulated by the level of odor in
the environment appears to be a generic component of the mechanisms used by
animals to locate resources using chemical cues. Animals using this mechanism
are not determining the direction of their locomotion according to the
chemical signal directly; rather, the chemical information is
modulating their orientation to flow information
(Bell and Tobin, 1982
;
Schöne, 1984
). As long as
the chemical signal is detected simultaneously with flow, the organism will
continue to orient and move counter to the direction of flow and toward the
source of the chemical (Baker et al.,
1984
; David et al.,
1983
; Baker and Haynes,
1987
; Willis and Cardé,
1990
).
Periplaneta americana, the American cockroach, has been studied
extensively as a model for odor-modulated behavior
(Rust and Bell, 1976;
Rust et al., 1976
;
Bell and Kramer, 1980
;
Bell and Tobin, 1981
;
Seelinger, 1984
;
Tobin, 1981
) and olfactory
processing (Boeckh et al.,
1987
). The majority of this previous work was performed on
tethered walking preparations (Rust and
Bell, 1976
; Rust et al.,
1976
) and animals free to walk but restricted to
computer-controlled locomotion compensators (Bell and Kramer,
1979
,
1980
), two similar but
unnatural conditions. Relatively little of the published work on the
odor-modulated orientation and navigation behavior of cockroaches reflects
research performed on animals moving freely through an environment in either
the laboratory (Bell and Tobin,
1981
; Tobin, 1981
)
or the field (Seelinger,
1984
).
These earlier experiments were aimed at determining the specific sensory
information and orientation mechanisms that could be used by walking
cockroaches to locate sources of odor in wind, or using odor information
alone. Specifically, these studies examined whether the changes in steering
observed during orientation to odor were caused: (1) by changes in the walking
speed or rate of turning in direct response to changes in odor concentration
(i.e. chemotactic orientation), or (2) by odor stimuli initiating the
production of pre-programmed internally stored steering maneuvers with no
direct relationship to the odor concentration (i.e. turning rate and direction
determined by internally stored information rather than directly correlated
with the environment; Bell and Tobin,
1982; Schöne,
1984
). Although it is easy to state this as a dichotomy, it is
probable that individual turning maneuvers executed by an organism during its
response to a specific environmental condition may result from either one of
these mechanisms or a combination
(Schöne, 1984
). Therefore
the orientation tracks observed may not be the result of a single orientation
mechanism, but rather the result of an individual switching amongst an array
of different alternative mechanisms, depending on the local environmental
conditions and the individual's internal state.
One of the few experiments in which freely walking P. americana
males oriented to wind and odor (i.e. female sex-attractant pheromone) was
aimed at determining whether the turns observed resulted from an ongoing
internal program of counterturning initiated upon odor contact or were
triggered by the decrease in concentration or loss of contact with pheromone
when the male encountered the clean air-pheromone boundary at the lateral edge
of the plume (Tobin, 1981).
Internally programmed counterturning is thought to be a primary mechanism
underling the zigzagging tracks generated during upwind orientation to odor in
flying moths (Arbas et al.,
1993
; Vickers and Baker,
1994
; Mafra-Neto and
Cardé, 1994
; Willis and
Arbas, 1997
). The results of Tobin's experiment
(Tobin, 1981
) showed that when
the distance between the plume's edges increased, the width of the males'
tracks increased in concert, with many of the observed turns occurring at the
edges of the time-averaged plume boundaries. These turns were interpreted as
resulting from a pre-programmed turning response to the loss of odor
(Tobin, 1981
;
Bell and Tobin, 1982
), in
contrast to an internal counterturn generator similar to that hypothesized for
moths (Kennedy, 1983
).
However, not all turns observed occurred at the time-averaged plume boundaries
and it was suggested that internally generated turns of various origins also
contributed to the final structure of the orientation tracks observed
(Tobin, 1981
).
The results presented here address the mechanisms underlying the olfactory
orientation behavior of P. americana males by challenging freely
walking cockroaches to orient and track different combinations of wind and
odor through an experimental arena in our laboratory wind tunnel. We use the
earlier experiments of Tobin
(1981) as a starting point. To
determine whether the walking male cockroaches tracking a plume of wind-borne
female pheromone were orienting directly to the olfactory stimuli, or whether
the olfactory information initiated and modulated orientation to the wind, we
observed the preferred orientation direction of males released in different
combinations of wind and the female pheromone component (-)-periplanone-B.
Once we had established the preferred orientation responses to wind and odor,
we tested whether males tracking pheromone plumes (1) turned in response to a
decrease in odor concentration or the loss of contact with odor caused by
encounters with the lateral margin of the time-averaged pheromone plume, or
(2) executed turns according to an internal counterturning generator. Our
working hypothesis was that the large concentration differences experienced at
the time-averaged edges of the odor plumes would be the primary stimulus
accounting for turns observed during plume tracking. Consequently, as the
plumes increased in width we expected the overall widths of the tracks to
increase in concert, as has been observed previously
(Tobin, 1981
). The goal of the
work presented here is to form the foundation of a series of experiments
examining the orientation mechanisms used by walking animals to track odor in
terrestrial environments, and to compare them explicitly to the more
extensively studied examples of odor plume tracking by flying insects,
especially moths.
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Materials and methods |
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Wind tunnel
We conducted experiments in low-light conditions with light levels of
approximately 7 lux provided by four voltage-regulated 25 W red incandescent
bulbs and one separately adjustable 25 W white incandescent bulb. There were
also seven 40-60 W infrared lights distributed around the working area to
provide illumination for the video cameras. Wind speed was set at 25 cm
s-1 (Tobin, 1981).
We released P. americana males onto to our experimental arena, a flat
aluminum platform (1.52 mx0.92 m) held 25.4 cm above the floor of a
Plexiglas wind tunnel with a 1 mx1 mx2.5 m working section. The
pheromone source holder was centered at the upwind end of the aluminum
experimental arena and it held the pheromone ca. 1 cm above the aluminum
platform. The odor plume was removed from the wind tunnel and the building
via an exhaust duct attached to the downwind end of the tunnel.
We recorded the responses of cockroaches to the different pheromone plume structures using 4 Burle TC355AC (Lancaster, PA, USA) B/W cameras: one positioned directly overhead with a field of view of 1.93 m long x 1 m wide; a second with a zoom lens positioned overhead with a field of view of 0.67 m long x 0.86 m wide; a third positioned on one side of the wind tunnel just above the level of the aluminum floor with a field of view of 0.44 m wide x 0.59 m tall; and a fourth placed at the downwind end of the wind tunnel with a field of view of 0.68 m wide x 0.20 m tall. These latter three cameras with smaller fields of view were all aimed and focused on a volume approximately 40 cm3 beginning 45.1 cm downwind of the pheromone source and ending 111.8 cmdownwind of the source. In all cases, we recorded the cockroaches' behavior at the standard frame rate for NTSC video of 30 frames s-1.
We used a Peak event and video control unit (Peak Performance Technologies, Inc., Engelwood, CO, USA) to synchronize the three cameras with smaller fields of view to enable the 3D reconstruction of individual antennal movements associated with plume tracking.
To determine the nature of the cockroaches' orientation movements with respect to the odor plume, we videorecorded titanium tetrachloride (TiCl4) smoke plumes issuing from the same size and shape filter paper sources used to dispense pheromone during odor-tracking experiments. The time-averaged plume boundaries were determined by digitizing and tracking the paths of smoke packets using our motion analysis system. Using this method we determined both the time-averaged crosswind width of the plume and the maximum time-averaged height above the floor of the experimental arena at five sampling points at different distances from the source: 0 cm from the source, 38 cm downwind, 76 cm downwind, 114 cm downwind and 152 cm downwind. Since the smoke plumes and the cockroach orientation behavior were recorded in the same conditions, we were able to overlay the time-averaged plume outlines and the animals' tracks to determine the association between the clean air-pheromone boundary and the cockroaches' tracking behavior.
Experimental design
We determined the characteristics of orientation with respect to the wind
in walking P. americana males by recording their behavior when
challenged with an experimental environment characterized by (1) no wind or
odor, (2) 25 cm s-1 wind with no odor, and (3) 25 cm s-1
wind carrying a plume issuing from a point-source bearing 0.1 ng of
(-)-periplanone-B. We released males from the center point of the experimental
arena to make any orientation direction available to them. For the purpose of
this analysis, we defined the orientation direction as the direction in which
the males were walking when they left the field of view of the camera. We used
3-18 week old (Seelinger,
1985; Abed et al.,
1993
) sexually mature virgin male P. americana for this
study, and placed them individually in cylindrical (3 cm tall x 10 cm
diameter) aluminum screen release cages in the darkened wind tunnel room at
the beginning of their scotophase, and left them to acclimate to room
conditions for 2 h prior to beginning experiments. The release cage dimensions
were important because males exhibited a reduced propensity to walk in the
treatments in which (-)-periplanone-B was absent; pilot studies showed that
shorter cages made it more likely that the males would leave the release
point.
We generated four different plume structures by varying the size, shape and
orientation of the pheromone source (Fig.
1). We generated the first plume structure by using a 0.7 cm
diameter circular filter paper disk (Whatman No.1, Eastbourne, East Sussex,
UK) held perpendicular to the airflow with an insect pin
(Fig. 1A). We used this source
size and shape to enable easier comparison to earlier studies of plume
tracking behavior in flying moths
(Cardé and Minks,
1997). [The majority of studies of moth flight orientation
behavior have used plumes issuing from point-sources of pheromone that are
either identical to (Willis and
Cardé, 1990
; Willis and
Arbas, 1991
; Charlton et al.,
1993
; Willis et al.,
1994
) or similar in size and shape to the point-source in these
experiments.] The structure of the second plume treatment was generated by
rotating the 0.7 filter paper disk 90°, so that the disk shape was
parallel to air flow in the wind tunnel, resulting in a very narrow plume
(modified after the `ribbon plume' of
Mafra-Neto and Cardé,
1994
) (Fig. 1B).
The third plume was meant to significantly increase the width of the plume
while nearly maintaining its concentration; this was achieved by increasing
the surface area of the source by ca. 25 times while also proportionally
increasing the dosage of pheromone solution applied to the source. The source
of this wide plume treatment was 14.3 cm wide x 0.7 cm tall
(Fig. 1C). The fourth type of
plume structure was generated by placing a Plexiglas cylinder (81.28 cm tall
x 7.62 cm diameter) 5 cm upwind of the 0.7 cm diameter circular filter
paper disk held perpendicular to airflow. This induces turbulence through
vortex shedding, which dramatically alters the plume structure
(Fig. 1D). We will refer to the
treatments as point-source plume, ribbon plume, wide plume and cylinder plume,
respectively, throughout this paper.
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In this experiment we placed the virgin P. americana males in larger release cages (13.5 cm tall x 5.7 cm diameter) to hold them at the downwind end of the pheromone plume for a 1 min pre-release period. The larger cages allowed the males more range of movement during the acclimation period and while waiting to be released during an experimental trial. For each experimental day, we randomized the animals used according to their age and each trial according to the order of treatment presentation (each animal was used only once). We introduced P. americana males to the respective plumes beginning 2 h into their scotophase and challenged the cockroaches to respond to the appropriate experimental conditions.
Pheromone
We used the same filter paper loaded with 0.1 ng synthetic
(-)-periplanone-B (Kitahara et al.,
1987; Kuwahara and Mori
1990
) for the point-source, ribbon and cylinder treatments. The
wide plume treatment was dosed ten times with this same amount pipetted evenly
across the surface of the filter paper, resulting in a source that bore 1.0 ng
of pheromone on ca. 25 times the area of the filter paper disks used for the
other treatments. This combination of size, shape and pheromone dosage
resulted in filter paper disk sources that were 0.25 ng cm-2 and a
wide filter paper source that was 0.1 ng cm-2. Earlier observations
by Tobin (1981
) and pilot
studies by us suggest that there is no observable difference in the pheromone
response of P. americana males in the range of
10-6-10-3 µg of (-)-periplanone-B
[(±)-periplanone-B in the case of
Tobin, 1981
].
Data analysis
We digitized the walking paths of male cockroaches tracking pheromone
plumes every 83 ms using a computerized motion analysis system (Peak Motus
7.1TM, Peak Performance Technologies, Inc.). The Motus software separates
each 1/30th s video frame into the two 1/60th s video fields that it
comprises. Therefore, the maximum temporal resolution from an NTSC video
recording is 1/60th s or 16.7 ms per sample. Thus, we digitized each 5th
position of the cockroach during its response to the experimental
treatments.
In the large field of view camera we digitized two points on the cockroach: the center of the head and the distal tip of the abdomen. In the three smaller field-of-view cameras we digitized four points on each animal: the distal tip of each antenna, the center of the head and the distal tip of the abdomen. In all cases the movement tracks that were quantified and presented in the figures were the path of the digitized point on the head.
The response variables we measured from the video-recorded cockroach tracks
were: track angle (orientation of the movement vector from one cockroach
position to the next with respect to the wind direction; due upwind is 0°
by convention), track width (distance between turn apices measured
perpendicular to the wind direction, as per
Kuenen and Baker, 1982),
groundspeed (cockroaches' walking speed measured from point to point along its
track), inter-turn duration (time between the apices of sequential turns), and
net velocity (net speed from the beginning to the end of each walking track).
For the purposes of our analysis a turn was the location at which the head
reached a local maximum or minimum value with respect to the lateral frame of
reference of the wind tunnel.
We also quantified the stopping behavior of males in different environmental conditions and the four plumes used in these experiments. The number and duration of stops made by each individual was recorded and grand means for each treatment group were compared in a manner similar to the rest of the experimental variables. For the purposes of this study an animal was said to be stopped if there was no movement between two sequential positions of the head point.
In addition we measured finer-scale kinematic variables including: body yaw
angle (angle of the longitudinal body axis as defined by a line drawn between
the head and the distal tip of the abdomen, with respect to the wind
direction), body pitch angle (angle of the body axis with respect to the
floor), inter-antennal distance (distance between the distal tips of the
antennae in three dimensions), and the height of the antenna above the floor
(measured from the distal tips of the antennae to the floor). Most of these
parameters, aside from those describing the kinematics of the antennae, have
been routinely measured from the performances of flying moths tracking
pheromone plumes (Kuenen and Baker,
1982; Willis and Arbas,
1991
; Arbas et al.,
1993
).
The experiment was designed as a randomized complete block design, with each experimental manipulation as a treatment, and a complete group of treatments was performed as a block each day. These data were analyzed using a general linear model (i.e. proc GLM) in SAS (ver. 8.2) to perform an analysis of variance (ANOVA) for a randomized complete block design. Treatments and individuals were randomized daily. Approximately five individuals were exposed to the first treatment, the treatment was then switched and the next five individuals were introduced sequentially, and so on. When an ANOVA revealed significant effects in the experiment, we applied a Tukey multiple comparison test to determine which track and kinematic variables differed significantly among the experimental treatments at the 0.05 (P<0.05) level.
According to our analysis there was an unexpected significant day effect
associated with certain response variables in our model. This day effect was
associated with only some of the track parameters measured (in the `wind plus
pheromone' experiment: track angle, body axis and inter-turn duration; in the
`plume structure' experiment: net velocity, groundspeed and inter-turn
duration) and the effect was observed on different days for each parameter,
therefore a single day could not be eliminated from the experimental design.
Because of this unexpected effect we plotted each track parameter by day and
treatment to determine any obvious patterns associated with days or order of
treatment presentation. The means of the variables associated with the
cylinder treatment stood out on all days and for this reason we removed it
from the analysis and repeated the analysis with only the ribbon, point-source
and wide treatments. The result of this analysis was that there is no effect
of the order of treatment presentation, and no predictable pattern in day
differences among variables. Thus, the cylinder plume treatment appeared to be
the main source of the treatment effect in the ANOVA results and we concluded
that the statistically significant day and day-by-treatment effects that we
observed were caused by incomplete randomization in the treatment-individual
groupings (Pilla et al., in
press).
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Results |
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The yaw angle of the cockroaches' body showed similar changes in the three treatments of this experiment and were similarly significantly different according to the changes from no wind, to wind, to wind plus pheromone (Table 1). The addition of wind and pheromone to the environment did not significantly affect the groundspeed at which the cockroaches walked, nor the overall width of their tracks (Table 1).However, the net velocity or speed from the point of release to the point at which the cockroach left the field of view of the camera increased significantly and incrementally as wind, and then wind and pheromone, were added to the environment (Table 1). By referring to the movement tracks (Fig. 2), it is clear that this increase in net velocity is a reflection of the increased `directedness' or polarization of the cockroaches' orientation once the wind and then the pheromone were added to the environment. The duration of the inter-turn intervals was significantly affected by the addition of pheromone but not by the addition of wind to the environment (Table 1). The inter-turn duration of males tracking a pheromone plume upwind was significantly less than males walking in no wind or wind of 25 cm s-1. There were no statistically significant differences in the average number of stops made by males in either wind, no wind or wind plus pheromone conditions (Table 1).Likewise, there were no significant differences in the durations of stops in any of the experimental conditions. The similarity in the stopping durations suggests that the stops we observed during walking orientation may be internally controlled sensory sampling or information processing periods.
The time-averaged boundaries of smoke plume visualizations of our four
experimental plumes, as viewed from overhead, are illustrated in
Fig. 1. Since the dimensions,
shape and material making up the source of the point-source plume is the same
as used in many previously published studies of odor-tracking behavior in
flying moths, it is particularly important for comparison
(Fig. 1A; Charlton et al., 1993). In
these experiments the point-source TiCl4 smoke plume was 2.4 cm
wide at the source and reached 7.7 cm wide at the downwind end of the
experimental arena. (Smoke plume measurements show that the same point-source
generates a plume 1.7 cm wide at the source and 6.2 cm at the downwind end of
the wind tunnel at the 100 cm s-1 wind speed often used in moth
flight experiments; Willis and Arbas,
1991
.) The time-averaged dimensions of the smoke plume issuing
from the ribbon source started narrower, at 1.5 cm, and remained narrower, at
6.1 cm, for the length of the experimental arena, than all other plumes tested
(Fig. 1B). At 17 cm, the
time-averaged dimensions of the plume issuing from the wide-source was ca. 7
times as wide as the point-source plume at the source and expanded to 26.5 cm
by the time it reached the downwind end of the experimental arena
(Fig. 1C). As expected, the
widest and most turbulent smoke plume issued from the point-source positioned
immediately downwind from a 7.6 cm diameter Plexiglas cylinder
(Fig. 1D). The plume downwind
of the cylinder was a turbulent wake of alternating vortices known as a vortex
street (Vogel, 1994
). These
vortices typically extended 81 cm from the floor of the experimental arena to
the top of the cylinder. According to Reynolds number and Strouhal numbers
determined for the experimental conditions in our wind tunnel, the vortex
shedding frequency for the cylinder should be ca. 0.6 vortices s-1
(Vogel, 1994
). The
time-averaged heights above the floor of the experimental arena that each
plume treatment extended to are also illustrated in
Fig. 1. The plume downwind of
the cylinder extended the furthest above the floor because the low pressure
area immediately downwind of the cylinder drew the smoke from the floor to the
top of the cylinder, and thus generated the widest and tallest plume used in
this experiment. The smoke plumes issuing from the other sources increased in
width and height as they moved downwind away from the source. In each of the
point-source, ribbon and wide plumes the height above the arena floor
decreased at the downwind end of the arena. This decrease was the result of
the close proximity of the exhaust, which removed the plumes from the wind
tunnel room.
The behavioral responses of P. americana males to these four
different types of plumes are illustrated in
Fig. 3. The three tracks in
each panel of Fig. 3 illustrate
the range of variability observed from our experimental population: one track
with the least turns (top track in each panel), one track with the most turns
(bottom track in each panel) and one track representing a typical response by
males to that treatment (central track in each panel). In general, the males
tracking the most turbulent plume, generated downwind of the cylinder
(Fig. 3D), had tracks whose
response variables were statistically different from the other three plume
treatments, and males tracking the point-source
(Fig. 3A), ribbon
(Fig. 3B) and wide plumes
(Fig. 3C) generated tracks
whose response variables were statistically the same
(Table 2). Males that tracked
the plumes generated downwind of the cylinder walked significantly slower, on
average, than males tracking any of the other plume treatments
(Table 2). The groundspeeds at
which males walked up the other three pheromone plume treatments were nearly
the same (Table 2). Average net
velocity measured from the males' performances showed a similar pattern
(Table 2). The average track
angles of males walking up the cylinder plume were significantly greater than
those steered by males in any of the other plume treatments
(Table 2). The explanation for
these large track angles is illustrated in
Fig. 3, which reveals that
males tracking the cylinder plume generated many more turns and steered their
tracks at angles much further off of the wind direction than most males in the
other treatments. Track angles steered by males in ribbon plumes were
significantly smaller, on average, than males tracking point-source plumes,
with males tracking wide plumes generating mean track angles that were not
significantly different from either (Table
2). Males walking upwind in the plumes generated downwind of the
cylinder steered the yaw angles of their bodies significantly more off of the
wind direction, on average, than males tracking the other plume treatments
(Table 2). There were no
statistically significant differences in the orientation of the yaw angle of
the body amongst males tracking any of the other plumes
(Table 2). The mean
orientations of the yaw angles of males tracking the four plume treatments in
our experiment were not equal to their mean track angle
(Table 2); that is, on average,
the direction that the males aimed their bodies was not the same direction as
the vector along which they moved (Fig.
3). In all cases the orientation of the body's yaw angle was, on
average, oriented more directly into the wind than the resulting walking
track. This sort of steering is also typical of male moths tracking female
pheromones in flight (Arbas et al.,
1993; Willis and Arbas,
1997
).
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Males tracking the four different plumes showed a similar pattern in their stopping behavior as observed in the other response variables (Table 2). There were no statistically significant differences in the number of stops observed from males tracking point-source, ribbon or wide plumes while those tracking the cylinder plumes stopped significantly more than males in any of the other treatments. There were no statistically significant differences in the duration of stops in any of the different plume treatments or in the treatments of the wind plus pheromone experiment (Tables 1, 2).
The temporal measurements of the pheromone tracking response showed trends similar to the parameters quantifying their speed and steering. The inter-turn durations measured from the responses of males tracking cylinder plumes were significantly longer than males tracking any of the other plume treatments (Table 2). The next longest were those measured from males tracking wide plumes, but these were ca. half the duration of males tracking plumes issuing from the cylinder (Table 2). Inter-turn durations of males tracking the wide plume were significantly greater than males tracking the ribbon plume, and the inter-turn durations of males tracking the point-source plume were not significantly different from either (Table 2).
It should be noted that the average groundspeeds exhibited by males tracking point-source plumes in the plume structure experiment and those of males tracking the point-source plume in the wind plus pheromone experiment were not as similar as one might expect. Even though the wind speed, pheromone source and plume structure were the same across these two experiments we observed the performance of the cockroaches in two different parts of the experimental arena and for different total distances and times. The male responses in the plume structure experiment were measured and averaged over the length of the entire experimental arena. In the wind and pheromone experiment the males were released at the lateral and longitudinal mid-point of the arena. These differences in the males' environment result in changes in their behavior. Males in the wind plus pheromone experiment were released ca. 75 cm closer to the pheromone source than those in the plume structure experiment. This would result in these males experiencing both different plume structure and a higher mean pheromone concentration than that present at the downwind end of the arena.
When we broke down the longer plume structure tracks lengthwise into three ca. 50 cm long sections (e.g. downwind, middle and upwind) and compared the average groundspeeds, we found that the males walked upwind more slowly in the area near the release point and as they approached the pheromone source, while they walked faster inbetween these two areas (data not shown). Thus, in the wind plus pheromone experiment we focused on the area near the release point where the males were walking more slowly.
On average, the antennae of P. americana are longer than the body length from head to the tip of the abdomen (antenna length, 4.8±0.3 cm; body length, 3.9±0.1 cm; means ± S.D.; N=5 individuals, i.e. 10 antennae). Since the antennae are the cockroaches' primary olfactory structure it was critical for the interpretation of the turning maneuvers observed to know where the antennae were during the odor-tracking performances. Our video recordings sub-sampled the plume tracking arena at higher spatial resolution to measure the position and angular orientation of the antennae with respect to the environment and the cockroaches' body in three dimensions. Fig. 4 shows examples of P. americana males tracking plumes from a point-source and a wide-source, with `exploded' views depicting the tracks of the tips of their antennae along with the body. This figure suggests at least two possible types of steering maneuvers executed by males during odor tracking: broad, gradual turns with continuous upwind progress (Fig. 4A), and temporally brief sharp turns, during which little or no upwind progress is made (Fig. 4B). Bytracking the positions of the tips of the antennae (broken lines in Fig. 4) it can be seen that an `envelope' of space much wider than the body is scanned for odor information. Furthermore, the distance between the tips of the antennae is held in a narrow range between 5 and 6 cm apart during pheromone tracking behavior, and this same limited range of inter-antennal distances is held by males tracking plumes from all four sources (P>0.05; Table 3). Thus, males maintaining their inter-antennal distances at or near the observed average values could theoretically walk straight upwind in point-source and ribbon plumes with the tips of their antennae protruding beyond the time-averaged plume boundaries into clean air for almost the entire length of the experimental arena. Such a scenario would have been impossible in the wide plume. Unless the cockroach approached close to the edge of the plume boundary (e.g. Fig. 4B), it could have tracked the full length of the wide plume while its body and antennae were completely imbedded in the pheromone plume. Likewise, in any of the four plume types, the height of the time-averaged plume envelope above the floor was always great enough that the probability of the tips of the antennae encountering the clean air-pheromone boundaries above them would have been very low (Table 3). In addition, there were no statistically significant differences in the average height at which the males held their antennae across the four different plume treatments (Table 3). Another way that males tracking the plumes could change the height of their antennae above the floor, while maintaining a stereotyped antennal posture, would be to alter the pitch angle of their body with respect to the floor (Fig. 5). By walking upwind while holding their heads higher above the floor, their antennae would consequently also be held further above the floor. However, the males tracking the plume downwind from the cylinder were the only individuals that showed behavior statistically different from the rest (Table 3).
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Similar measurements were made describing the antennal behavior of males orienting in different wind and pheromone conditions, with similar results (Table 4). As with males orienting to different plumes, males in the different wind and pheromone conditions showed essentially the same inter-antennal distances, with almost the same average maximum and minimum angles displayed by the males tracking the four different plumes (Tables 3, 4). The small differences between the mean inter-antennal distances of the males in wind and pheromone (Table 4), and those generated by males tracking plumes (Table 3), could be the result of the shorter length of track measured in the orientation experiment (due to the smaller field of view recorded) and the position of the recording area in the larger experimental arena (see discussion of differences between these experiments, above). We expected to observe greater differences between males in zero wind and zero pheromone and males experiencing environmental conditions including wind and odor, but instead found their antennal behaviors to be broadly similar both in inter-antennal distance and height above the floor (Table 4). The mean pitch angles of the body adopted by males orienting to wind alone were significantly less compared to males orienting to wind and pheromone and zero wind and zero pheromone (Table 4). Further fine-scaled analysis of the association of specific antennal postures and movements, tracking behavior and plume structure from this data set is in preparation and will be published separately.
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Discussion |
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Previous work on the orientation of walking with respect to the wind
direction showed that P. americana males walking on a locomotion
compensator preferentially turned into the wind and walked upwind at wind
speeds below 20 cm s-1 (Bell and
Kramer, 1979). When the wind speed increased above 20 cm
s-1 they turned and walked with the wind direction, downwind
(Bell and Kramer, 1979
). These
results demonstrated that walking cockroaches show a positive or negative
anemotactic response depending on the wind speed. Our experimental design did
not include exposure to wind speeds between zero and 25 cm s-1, so
our results cannot speak to this earlier observation. As in our experiments,
when female sex pheromone was added to the air stream the male cockroaches
exhibited an odor-mediated change in their orientation preferences, changing
from orienting with the wind direction to orienting into the wind
(Bell and Kramer, 1980
). Thus,
the results of our experiments with freely walking cockroaches support and
extend the results of the earlier work using restrained preparations.
One important difference between our results and the earlier experiments
studying cockroaches and the small beetle Trogoderma variable, is
that insects in these earlier experiments oriented to wind and odor on a
servosphere apparatus by walking upwind at an angle to the wind
(Bell and Kramer, 1980;
Tobin and Bell, 1986
). In
neither case was a stereotyped orientation angle steered with respect to the
wind. However, each individual's mean steering angle was consistent across
trials. P. americana males in our experiments showed no such tendency
for off-axis orientation to the wind, and in most cases showed an overall
trend to walk directly upwind while in the pheromone plume (Figs
2,
3). One obvious explanation for
this difference in behavior is the different arenas used in these experiments.
Males walking in our laboratory wind tunnel move upwind through a visual
environment consisting of a variety of objects (e.g. camera mounts, infra-red
lights, structural components of the wind tunnel, etc.) oriented at variable
angles with respect to their horizon. Most diagrams and photographs of
servosphere locomotion compensators show what appears to be a uniformly sparse
visual environment (Tobin and Bell,
1986
; Kramer,
1976
). In addition, these experiments are typically described as
being performed in total darkness (Tobin
and Bell, 1986
).
It has been demonstrated that P. americana orient their walking
orientation with respect to visual landmarks
(Mizunami et al., 1998) and
that the addition of fixed visual cues to studies of the walking orientation
of female crickets to auditory stimuli (male calling song) on servosphere
locomotion compensators resulted in tracks that were oriented more directly
toward the sound source (Weber,
1990
; Böhm et al.,
1991
). Thus, steering with respect to the visual surroundings may
explain some of the differences in behavioral observations of odor-modulated
upwind walking in P. americana between studies using locomotion
compensators and our wind tunnel, but this awaits future experimentation.
Effects of plume structure on olfactory orientation behavior
The responses of P. americana males to the different plume
structures presented in our experiments can be placed in two general
categories: one comprising the performances of males responding to the ribbon,
point- and wide-source and the other comprising those responding to the
cylinder plume (Table 2,
Fig. 3). Some individuals
responding to the ribbon, point- and wide-source plumes walked upwind in the
plume, generating tracks that often had segments aimed nearly directly upwind
in the plume, while the tracks of others showed `moth-like' examples of
temporally regular zigzagging counterturns
(Fig. 3). A few individuals
showed examples of relatively sharp `turns-back' at or near the time-averaged
boundaries of the odor plume that could be interpreted as being caused by
encounters with the abrupt clean air-pheromone edge at the lateral margins of
the plume (Figs 3,
4). The males responding to
plumes in the turbulent wake of the cylinder also generated tracks with
segments of zigzagging turning made up of temporally regular counterturns
together with irregular sharp rapid turns, suggesting turning back at a
distinct clean air-pheromone edge. However, because of the highly turbulent
nature of the cylinder's wake and the possibility that the males responded to
the large turbulent eddies as apparent shifts in wind direction, the responses
of males tracking the cylinder plumes were significantly more variable than
those tracking any of the other plume treatments
(Fig. 3).
If our P. americana males had behaved according to our working
hypothesis, their plume tracking paths should have increased in overall width
as the plumes that they were tracking increased in width. In a limited sense
that is what we found (Table
2), but our results are not consistent with those published
previously (Tobin, 1981). By
adding the ribbon and cylinder plume treatments to our experiment we
challenged our males to track plumes that were both narrower and wider than
those used by Tobin (1981
).
The ribbon plume could have elicited narrower orientation tracks for at least
two reasons. First, the source of the ribbon plume was so narrow (0.5 mm) that
no turbulent plume was generated (our TiCl4 smoke plume
observations confirmed this - no turbulent eddies were generated downwind of
the ribbon plume source). This lack of turbulence meant less mixing between
the pheromone-bearing ribbon and clean air, resulting in a plume that was at
least slightly more concentrated. The plume's narrow cross section also means
that, if the same number of molecules evaporated off the ribbon source as
evaporated off of the point-source, those molecules were now occupying a
smaller volume, resulting in a higher concentration. Increased odor
concentration has been shown to cause male moths tracking plumes in flight to
generate narrower tracks than those tracking plumes of relatively lower
concentrations (Kuenen and Baker,
1982
; Charlton et al.,
1993
). Thus, it is possible that the higher pheromone
concentration experienced by the males tracking the ribbon plume could have
affected the overall width of their walking tracks. In only one case have
different source concentrations of pheromone been presented to walking
cockroaches, and in that study P. americana males generated
straighter tracks while walking upwind in plumes issuing from higher
concentration sources on a locomotion compensator
(Bell and Kramer, 1980
). The
second point to consider is that the width of the time-averaged plume envelope
of the ribbon plume would have allowed a male positioned near the plume's
centerline to walk upwind with at least the distal parts of both antennae
extending through the lateral margins of the plume into clean air
(Fig. 3,
Table 3). Thus, if P.
americana males can make spatial comparisons between their two antennae,
these males could have been able to position themselves in the plume and
maintain upwind progress with few or only minor turning maneuvers. Visual
inspection of the significantly narrower movement paths
(Fig. 3), and inter-antennal
distances that span the time-averaged plume envelope (Tables
2,
4) support this idea. Of course
both of the above mechanisms, or others, could have influenced the
cockroaches' steering maneuvers simultaneously.
The biggest difference between the results presented here and the earlier
results (Tobin, 1981) was
observed in the comparison between the male responses to our point-source and
wide plumes. The turbulent structure of these two plumes should have been very
similar because the wind speed in these experiments was the same, and the
diameter of the point-source was equal to the height of the wide-source. The
combination of these facts should have resulted in the shedding of turbulent
vortices in the same range of sizes and frequencies. Our direct visual
observations of TiCl4 smoke plumes support this. Thus, with the
turbulent structure and the amounts of pheromone/area of these two sources
being very similar, the main difference between these two treatments was the
width of the plume. According to our working hypothesis and the previously
published data (Tobin, 1981
),
this should have resulted in males generating orientation tracks with
substantially different structures. However, this was not the case. Whether
comparing the quantitative measurements
(Table 2) or visually assessing
the qualitative aspects of the tracking performance
(Fig. 3), it is clear that the
responses of P. americana males to the point-source and wide plumes
under our experimental conditions were essentially the same. Thus, we must
conclude that the abrupt spatial and rapid temporal decrease in pheromone
concentration at the lateral time-averaged plume boundaries did not account
for the width of the walking tracks we observed from males tracking the wide
plume to the source. However, it is certainly possible that males responding
to the point-source plume could have tracked the plume in a manner similar to
that proposed above for the ribbon plume, by spatial comparison of the distal
tips of the antenna projecting beyond the time-averaged plume boundaries.
One must also remember that the turbulent nature of the pheromone plume means that males tracking all but the ribbon plume would have been experiencing a rapid and continuous stream of pheromone onsets and offsets; truly an intermittent pheromone stimulation. Furthermore, the time-averaged plume boundaries are an artifact of sampling and plume statistics and a fixed line between pheromone and clean air does not exist. However, the relative difference, both spatially and temporally, between offset and onset of pheromone at the plume boundaries would be expected to be greater than those experienced within the plume envelope.
Antennal structure and behavior
The ability of P. americana to utilize the spatial distribution of
odors to control its steering maneuvers and orientation is critical to our
understanding of this behavior. It is clear that the long, maneuverable
antennae of these animals could provide information on the spatial
distribution of odor as much as a body length away from the head on both
sides. In earlier work it was not known whether P. americana males
averaged the chemical information detected across the whole antennae, or if
they could detect odor stimuli impinging on specific segments along the length
of the antennae (Tobin,
1981).
While it is still not known whether the sensory processing centers in the
brain of P. americana average odor information across the entire
length of its antennae, it is known that there is a substrate in the central
nervous system that could enable the cockroaches to determine where along the
length of their antenna an odor stimulus exists. The antennal lobes of male
cockroaches P. americana, and moths Manduca sexta, have been
shown to possess projection neurons that receive inputs from olfactory
receptor cells from spatially distinct locations along the length of the
antennae (Hösl, 1990;
Heinbockel and Hildebrand,
1998
). The response properties of these neurons indicate that the
higher centers of the brains of male cockroaches receive information that
should enable them to discriminate between odor contacts made at the proximal
end, the middle and the distal tip of their antennae
(Hösl, 1990
). This system
could have provided the information on the spatial distribution of the
pheromone plume necessary to enable the males in our experiment to walk
directly upwind along a plume that was narrow enough for part of their
antennae to be in the plume while part projected into clean air. Similar types
of projection neurons with spatially distinct receptive fields have also been
identified in the antennal lobes of male moths
(Heinbockel and Hildebrand,
1998
), but their potential role in supporting olfactory
orientation in flight or walking is much less clear.
The distribution of pheromone-sensitive sensilla along the length of male
cockroach antennae has also been studied
(Schaller, 1978). Based on
mainly morphological data, the study showed that putative pheromone-sensitive
hairs composed different proportions of the population of sensilla in
different parts of the male antenna. Pheromone-sensitive sensilla made up ca.
80, 50 and 30% of the sensillar population in the proximal, medial and distal
segments of the male antenna respectively
(Schaller, 1978
).
Pre-programmed behavior during olfactory orientation
What adaptive advantage might the execution of an internally generated
program of counterturns provide to a P. americana male walking upwind
in a plume of female pheromone? It has been argued repeatedly that the
temporally regular counterturning observed in flying male moths tracking
pheromone plumes is an active sampling behavior serving to expose the moths to
wind-induced drift of the visual flow-field information necessary to detect
any differences between their intended flight path and the path that they
actually fly along. This exposure to being drifted off course thus enables
rapid and more precise adaptation to changes in wind speed and shifting wind
directions (Kennedy, 1983;
Cardé, 1984
;
Baker, 1985
). However, its
primary function has always been thought to increase the volume of air scanned
by the moth in order to remain in contact with, or reacquire, an elusive
olfactory stimulus (Kennedy,
1983
). Walking male cockroaches, like any walking insect, should
not require the sort of active scanning for wind information argued for the
flying moths. Since they are in constant contact with the ground they should
have constant and perfect information on the direction and magnitude of air
flow, as provided by mechanosensory hairs and specialized wind sensing organs
on the antennae (Bell and Kramer,
1979
; Gewecke,
1977
). In fact, when moths that normally fly upwind to female
pheromone are forced to walk upwind to the same source, the temporally regular
counterturns characterizing flight tracks are not observed
(Willis and Baker, 1987
).
Alternatively, intermittent expression of an internal program of counterturns
could serve the scanning function proposed by Kennedy
(1983
). A modified version of
Kennedy's idea has been proposed by Tobin and Bell
(1986
) to explain the
zigzagging walking tracks of a small beetle walking upwind to their female
pheromone. Tobin and Bell
(1986
) suggest that males
executing internally generated counterturns would be limiting the lateral
extent of their walking tracks, and thus maintain contact with the odor plume.
This might be especially important while tracking a pheromone plume in the
shifting wind and complex environments encountered by male cockroaches in
their natural environment.
Acquisition of olfactory information via specific behavioral adaptations may also be occurring during odor-tracking behavior in P. americana males. At some point in the responses of many of the males, we observed individuals that stopped during their orientation tracks (Tables 1, 2). There are many possible explanations for stopping during upwind odor-tracking behavior. Males could be stopping to sample or actively scan the environment to update their information on wind, odor or other variables. What we observed as a stop could have been the male experiencing a decrease or increase in odor concentration large enough to trigger the beginning of a shift in behavior from tracking to searching behavior. Another explanation could be that stopping or slowing down is a requirement for executing sharp turning maneuvers, i.e. the turn to be executed is large enough and the duration short enough that the male must slow down in order to successfully perform the maneuver. While the latter explanation is almost certainly true, we observed stopping behavior that was not associated with turns (data not shown). The number of males that stopped during odor tracking in different plumes might shed light on the role of stopping during odor tracking. Few of the males tracking the point-source and wide plumes stopped (Table 2), while more than half of the males tracking the ribbon plume and all of the males tracking the cylinder plume stopped sometime during their performance (Table 2). One possible interpretation of this result is that more individuals stopped in the two treatments in which males might have most easily experienced a loss of contact with the plume. The loss of plume contact in males tracking the ribbon plume could have been caused by their own maneuvering i.e. the plume was narrow enough so that relatively small magnitude turning could have taken an individual out of contact with the plume or resulted in bilaterally asymmetrical stimulation of the antennae. It is easy to imagine the turbulent vortex street downwind of the cylinder resulting in repeated loss of odor contact during tracking. Interestingly, males tracking the two plume treatments with the most similar temporal/spatial filamentous structure (i.e. the point-source and wide plumes) had similar mean numbers of stops, and the lowest numbers of individuals stopping during odor tracking (Table 2). Thus, the relatively rapid and continuous pattern of pheromone onset and offset experienced during contact with the point-source and wide plumes may be a `better' stimulus resulting in the ongoing expression of upwind tracking and walking. Taken together, these two results suggest that rapid, large scale changes in olfactory environment may trigger or contribute to the expression of stopping during upwind odor-tracking behavior in P. americana. In our study, more P. americana males released into an environment in the absence of wind and pheromone stopped than most of the males in the plume structure experiment (Table 1), suggesting that stopping while walking may be a generic sampling behavior, or response to large or rapid environmental changes of any kind.
It is also interesting to note that in both experiments reported here there
were no significant differences in the duration of the stops produced by
individuals in any of the experimental treatments. The homogeneous nature of
the stop durations in these different environmental conditions suggests that
stopping during walking may be a preprogrammed behavioral response. Similar
stereotyped stopping behavior has been observed in female frogs using their
ears to orient and navigate toward male calling songs
(Rheinlaender et al., 1979).
However, while stopped, these female frogs actively scan their heads from side
to side, adjust their steering direction, and continue walking. A detailed
analysis of the stopping behavior of our male cockroaches and associated
active scanning behavior is ongoing and will be published in the future.
Temporal structure of olfactory orientation behavior
We measured the timing of the turning behavior observed from walking P.
americana males in the same manner used to analyze the upwind flights of
male moths tracking plumes of pheromone
(Willis and Baker, 1987;
Willis and Arbas, 1991
). In
most cases the average inter-turn durations measured from walking male
cockroaches in this experiment were similar to those measured from flying
Manduca sexta males tracking pheromone plumes in a laboratory wind
tunnel (Table 2; see table 1 in
Willis and Arbas, 1991
). The
inter-turn durations in M. sexta are the most stereotyped aspect of
their odor-tracking behavior known. So far the only experimental manipulation
known to cause flying M. sexta males to change their inter-turn
durations is removing the odor source during upwind orientation, thus forcing
the males to change from tracking an odor plume to flight in clean air
(Willis and Arbas, 1991
). The
stereotyped nature of the turn timing in pheromone-tracking flying moths has
led researchers to hypothesize the existence of a central nervous system turn
timer that is activated during pheromone-tracking behavior
(Kennedy, 1983
;
Willis and Arbas, 1997
). Such
a timer has also been hypothesized to underlie at least some of the turning
maneuvers executed by plume tracking P. americana males walking
upwind to a source of female pheromone
(Tobin, 1981
). Males tracking
pheromone plumes in our experiments generated sequential series of
counterturns that appeared very similar to the plume following tracks of
flying moths (Fig. 3). However,
the variability of the timing of the turns in our walking tracks is an order
of magnitude larger than that measured from the tracks of male moths flying
upwind in plumes of pheromone (Table
2; see table 1 in Willis and
Arbas, 1991
). This difference in the variability in turn timing
may reflect the differences in the two modes of locomotion, specifically the
lower speed of walking and thus the increased ability to respond to fine scale
changes in the olfactory stimulus.
By making explicit comparisons between the relatively well-studied odor-tracking behaviors of male moths, and walking odor trackers, we expect to gain insights into both systems. Clearly the intermittent plumes encountered by both walkers and fliers are shaped by the same sort of environmentally determined turbulence, and they must perform similar odor-tracking tasks to locate the source. By comparing how these different odor trackers have been shaped by similar environmental constraints, and the effects of different locomotory and sensory behaviors, we may reveal core similarities and unique adaptive solutions to the complex task of locating distant unseen odor sources.
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Acknowledgments |
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