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First published online February 15, 2008
Journal of Experimental Biology 211, 757-765 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.012773
Finding females: pheromone-guided reproductive tracking behavior by male Nereis succinea in the marine environment
1 Department of Physiology, Wayne State University, Detroit, MI 48201, USA
2 Department of Biological Sciences, University of Hull, Hull HU6 7RX, UK
3 Department of Computer Science, Wayne State University, Detroit, MI 48202,
USA
* Author for correspondence (e-mail: jeffram{at}med.wayne.edu)
Accepted 11 December 2007
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Key words: Annelida, computer simulation, cysteine-glutathione disulfide, polychaete, reproduction
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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) can
facilitate fertilization; however, such gamete-level mechanisms are helpful
only over short distances (<1 mm, the distance sperm may swim before being
dispersed by bulk flow or turbulence). Of greater importance is making sure
that males release gametes in close proximity to the females so that gamete
interactions have a chance to occur. Chemical attractants (pheromones) may
play a role in attracting mates to one another. However, an important gap in
our knowledge is that not many marine pheromones have been characterized
chemically, and of those that have been characterized the pheromone-release
and stimulus–response patterns that mediate close encounters have not
been described. The marine worm Nereis succinea Frey and Leuchart
1847 (Annelida, Polychaeta, Aciculata, Nereididae; synonyms: Neanthes
succinea and Nereis limbata), which is well known for its
dramatic reproductive swarming behavior, is an excellent model system in which
to investigate the mechanisms that mediate pheromone-guided reproductive
behaviors (Hardege, 1999). Not
only is its behavior easily observable in the field
(Costello and Henley, 1971) and
in the laboratory (Ram et al.,
1999), but also the structure of the female pheromone that
triggers male spawning (cysteine glutathione disulfide, CSSG) has been
determined (Zeeck et al.,
1998b) and is readily synthesized and isolated
(Hardege, 1999).
N. succinea ripens into mature male and female heteronereids while
residing in sediments of brackish tidal areas and emerges to spawn,
synchronized by the influence of circadian, lunar and annual cycles
(Lillie and Just, 1913). N.
succinea spawn only during summer months, especially around the new moon
or full moon, and for just a few hours beginning shortly after dusk. Upon
emergence from the sediments, male and female worms swarm (swim actively) at
the water surface, searching for the opposite sex. If male and female N.
succinea approach each other within about 3 cm they exhibit a `nuptial
dance' in which males and females circle rapidly around one another releasing
clouds of sperm and eggs. Following the nuptial dance, males swim off in
search of other females, while the females, having released all of their
oocytes, stop swimming and appear to die, as they sink from the surface.
The nuptial dance and gamete release behaviors of N. succinea are
triggered by sex-specific pheromones, with sperm release being triggered in
males by CSSG excreted by females (Hardege,
1999; Hardege et al.,
2004; Zeeck et al.,
1998b), and a bouquet of inosine and amino acids released by males
with the sperm (Zeeck et al.,
1998a) triggering egg release in females. CSSG has the threefold
effect on males: arousing swim speed, activating circling behavior, and
rapidly eliciting ejaculation (Ram et al.,
1999). However, an important part of this encounter that has not
been worked out is what mechanisms mediate the approach within 3 cm to enable
the pheromone-mediated nuptial dance to occur.
Although the density of some swarming polychaete species may be high enough for encounters to occur by random chance, additional mechanisms may facilitate these encounters in N. succinea. The use of mechanisms other than random encounters in N. succinea is suggested by observed N. succinea swarming densities, anecdotal observations of N. succinea swarming behavior, and quantitative observations of swimming speeds and CSSG release by swarming animals. The appearance of a female on the surface, recognizable by its greener color (Fig. S1 in supplementary material) and slower swim speed than males, has often been followed by a seemingly higher density of males than before and a resultant nuptial dance and spawning, suggesting that males somehow sense females from a distance, are attracted to them, and can track them down.
By what mechanism might males detect the presence of females and how might
these cues mediate the close approach necessary for spawning to take place? In
this paper we propose the hypothesis that the pheromone CSSG has a dual
function, with low concentration pheromone trails being used by male N.
succinea to find females and increase their likelihood of mating, whereas
high concentrations of the same pheromone trigger the spawning behavior
itself. The spawning function of relatively high concentrations of CSSG
(
10–7–10–6 mol
l–1) is already well established
(Ram et al., 1999), so we have
focused here on the hypothesized search behavior in response to lower
concentrations of pheromone. For behavioral experiments, we devised a unique,
heart-shaped aquarium to facilitate observations of male worm responses to
linear pheromone trails. Computer simulation was then used to show that male
responses to low concentrations of pheromone can increase the frequency of
finding female N. succinea on pheromone trails.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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57% seawater), and fed with
Tetra MarineTM, frozen mussel and live Corophium volutator twice
a week. Moonlight was simulated with a 4 W lamp for four consecutive nights
every month (Hardege, 1990).
Animals that emerged and began swimming, indicating their ripening into mature
animals, were removed and stored in separate containers in DSW at 8°C
until use. Under such conditions, ripe male N. succinea resume active
swimming upon warming to ambient temperature (22°C) and are responsive to
stimulation by CSSG. Any animals not swimming spontaneously within 30 min of
re-warming were discarded.
Behavioral testing
Initial tests were conducted in a standard rectangular aquarium filled to a
depth of 1.0 cm; however, worms often swam almost all of the time along the
walls and would get `stuck' in corners (head-in, with continued swimming
motion) and only relatively infrequently head out into the open field where
they might encounter the stimulus trails. Animals also usually swam along the
periphery of round tanks. Accordingly, we designed a unique `rounded
heart-shape' test apparatus (Fig.
1; see also Movie 1 in supplementary material), which had no
corners, so that upon reaching the internal apex, animals swimming along the
wall would be directed into the open field. The test apparatus was filled to a
depth of 1.0 cm with DSW.
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). All
solutions contained a dye [0.03% fluorescein sodium salt or black ink (Parker
Quink)] that preliminary experiments showed had no behavioral effects at the
concentrations used. Trails were often laid down perpendicular to and just
below the internal apex of the heart apparatus
(Fig. 1 and subsequent data)
but occasionally at an oblique angle or criss-crossing one another. A typical
trail was created by ejecting approximately 0.2 ml of solution to produce a
pheromone plus dye trail approximately 20 cm long. Trails were 5–10 mm
wide, judging by the spread of the dye. Assuming an approximately cylindrical
trail of diameter 5–10 mm and length 20 cm (a volume of 4–16
ml), then the solution in the syringe is diluted 20- to 80-fold in the
pheromone trail. Since the exact dilution is unknown, concentrations are given
in the text as `estimated' () concentrations, and the exact concentration
in the syringe is given in figure captions to make the source of the estimate
apparent. Behavioral responses were recorded by video, digitized, and then played back frame-by-frame with Sony Movie Studio, plotting animal positions every 0.2 s.
Simulation modeling
The movement of male worms was modeled in two dimensions with respect to
swim speed, turns, and the effect of encountering a chemical stimulus on these
variables. The chemical stimulus was modeled as a linear trail of given
concentration and width. To simulate mechanical dispersion and mixing of the
chemical by the modeled worm swimming through it, the chemical at a particular
location could be dispersed into adjacent locations with no loss of total
chemical, thereby reducing its concentration in its original location and
spreading it to adjacent areas. The amount of dispersion caused by the modeled
worm was a user-adjustable variable. In the simulation, the movement of the
worm over a time, dt, is determined according to the swim speed and
turning angle calculated from pheromone concentration–response and
behavioral arousal curves described in the Results; the worm is then moved to
the new calculated position, and the process is repeated, keeping track of the
worm's previous positions, turning angle and speed. The simulations in this
paper used a dt of 50 ms. The model also includes small spontaneous
direction changes that occur according to whether a random number generated
each `dt cycle' exceeds a value set by the user. Sources of
quantitative values (swim speeds, turning, CSSG sensitivity) are given with
the model, in the Results.
The simulation was programmed in JAVA and has been placed on the internet for testing and general access. The URL is http://paris.cs.wayne.edu/~aw6056/Simulation/Simulation.html.
Since the model disperses the chemical in the trail, the length of trail over which the simulated chemical was dispersed (i.e. of lower concentration after passage of the simulated worm) was measured. For simple crossing of the trail, this would be the width of the path through the trail. Where the simulated worm traveled along the trail for some distance, this is the contiguous length of simulated trail disturbed by the simulated animal before veering off in another direction.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Fig. 1 illustrates representative swim tracks of male worms with no pheromone trail present (Fig. 1A), a pheromone trail present in a distant part of the tank (Fig. 1B), or a pheromone trail present at a very low concentration (Fig. 1C; <10–9 mol l–1, estimated as described in Materials and methods). In summary, spontaneous swimming behavior of N. succinea is characterized by (1) frequently swimming along edges, (2) when swimming in the open field, swimming straight (e.g. Fig. 1C), or (3) when turning in the open field, swimming in broad, gradual curves with occasional changes in direction.
Deflection and trail-following upon encountering a pheromone trail
With a moderately higher concentration of pheromone, the usual response of
male worms upon encountering the trail was a deflection of the swim path
(Fig. 1D). The deflection
occurred about equally often to the left as to the right. For nine animals
crossing trails at relatively low concentrations (CSSG between
10–9 mol l–1 and 10–8 mol
l–1 or glutathione 10- to 100-fold higher), deflections of
the swim path averaged 44±7% to the left, 39±6% to the right and
16±3% no turn (mean ± s.e.m., N=9 animals).
When crossing a low-concentration trail at right angles the deflection was usually not large enough to bring the stimulated worm back to the trail. However, encounters at an oblique angle often resulted in encountering the trail again, which again deflected the direction of swimming. The resultant `weaving' behavior by a representative animal is illustrated in Fig. 2.
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Circle-swimming along the trail
Finally, when male worms encountered pheromone trails at high enough
concentrations that the worms were stimulated to turn at least a semicircle to
bring them back to the trail, the animals exhibited extensive circling
behavior that resulted in relatively long sections of the pheromone trail
being encountered. In the example illustrated in
Fig. 4, the male worm
immediately began circling and speeding up upon encountering a trail at a
concentration above 10–8 mol l–1. Prior to
encountering the pheromone, the worm had been swimming along the aquarium wall
at a moderate speed (85 mm s–1;
Fig. 4A) and with some
hesitation. Fig. 4B,C shows how
the pheromone stimulated rapid circling. A further effect was that the worm
dispersed the trail by swimming through it multiple times, so that eventually
the worm responded to the decreased concentration of pheromone with larger
circles and occasional changes in the direction of circling
(Fig. 4D). However, when the
worm again swam into undisturbed pheromone at the left end of the trail
(Fig. 4E), it resumed swimming
in the smaller circles characteristic of the responses of male worms to higher
concentrations of pheromone. Eventually, the trail became so dispersed that
the worm swam through it without making a sustained turning movement, and thus
swam away from the trail with, a now, aroused higher swimming speed (170
mm s–1; Fig.
4F). The behavior of this animal in response to the pheromone
trail is also shown in Movie 1 in the supplementary material. Typically,
animals encountering pheromone trails at this concentration circled back to
the trail an average of seven times (±1 time, N=17 instances)
and usually encountered 10–20 cm of pheromone trail before swimming
away.
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Thus, laboratory experiments demonstrate that the turning and circling responses of male worms to pheromone encountered in linear trails of the chemical stimulate worms to encounter more of the trail. In order to determine whether this behavior would help male worms encounter females that could have produced such trails, the behavior was simulated in a computer model.
Simulation parameters
Parameter values for modeling the sensitivity, rate and duration of arousal
of the circling and swim speed behavior of male worms in response to pheromone
were determined from information in previous publications, as described below,
in order to avoid `circular reasoning' that would result from choosing
parameter values from the behavior to be modeled. Dimensions of the pheromone
trail were chosen on the basis of previous measurements of the rate at which
females excrete pheromone, and the assumption that the trail was a cylinder of
uniform concentration and width that would become dispersed as a result of
mixing when animals swam through it. The model was used initially to see if
that information alone would yield the trail-following behavior seen in the
above experiments. Subsequently, the model was tested for effects of pheromone
on the frequency of simulated male encounters with simulated females.
Responses of males to CSSG
Accelerated swim speed
CSSG arouses the swim speed of male N. succinea to two- to
fourfold pre-arousal speeds. Ram et al.
[(Ram et al., 1999) see
fig. 4] showed that a
representative male accelerated from 50 mm s–1 to
180±30 mm s–1 (mean ± s.d., average speed
3–5 s after CSSG stimulation) in response to a spawning dose
(10–6 mol l–1) of CSSG. Similarly, Ram and
Hardege [(Ram and Hardege,
2005) see fig. 2)]
showed that a male accelerated from 80 mm s–1 to 200 mm
s–1 in response to 10–6 mol
l–1 CSSG.
The threshold concentration for swimming arousal is one to three orders of
magnitude lower than needed to induce spawning. In Woods Hole, males that did
not spawn until stimulated with 10–6 mol l–1
CSSG exhibited accelerated swimming (a bit less than doubling the speed) at
10–7 mol l–1
(Ram and Hardege, 2005). At
Isefjord, males were more sensitive, showing 30–40% increases in swim
speeds in response to 10–10 mol l–1 CSSG
(Hardege et al., 2004) and
significant 10–20% increases in response to 10–10 mol
l–1 CSSG (Ram and Hardege,
2005). Threshold concentrations for arousal by CSSG varied among
animals collected from different locations (J.D.H., unpublished observations).
In the model that we illustrate, we used a `standard'-looking
dose–response curve that had a reasonably good match to the previous
experimental data: a baseline speed of 50 mm s–1, a 12%
increase in speed in response to a concentration of 10–9 mol
l–1, a doubling of speed at 10–7 mol
l–1 and a 2.6-fold increase in speed at 10–6
mol l–1 (Fig.
5A).
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) also
showed that in response to a high concentration of CSSG, male swim speed took
4 s to accelerate from 50 mm s–1 to 180 mm
s–1 and then slowed to its original level over the next
minute. Loss of swimming arousal over the course of about 1 min was also
reported for a second animal. The corresponding time course of arousal and
relaxation of N. succinea swim speed in response to CSSG in the model
is illustrated in Fig. 5B.
Circling behavior
Circling behavior and aroused swimming speed are independent components of
the response to CSSG, with the accelerated swimming out-lasting the circling
behavior in time. Ram et al.'s fig.
4 (Ram et al.,
1999), showed that upon encounter with pheromone, the male worm
initially made a semi-circle 10 mm in diameter with little increase in
speed, and then as the worm accelerated, swam in a circle 20 mm in
diameter. The angle turned per second was 500° s–1,
with the larger diameter circle resulting mainly from the increased speed.
Upon leaving the area of the dish in which the pheromone was applied, the
animal's speed was maintained at a highly aroused level while the worm curved
its swimming path only as much as required because of the perimeter of the
dish. Upon re-entry into the `cloud' of CSSG, the worm resumed circling, with
a rate of 600–700° s–1. A video of another animal
(http://sun.science.wayne.edu/~jram/N_succinea.htm),
shows similar behavior with a maximum turning rate in response to CSSG of
625° s–1. Previous experiments have not looked
closely at turning angles for lower concentrations of pheromone, so we have
simply assumed a similar dose–response curve to the one used for speed
(Fig. 5C). This assumption
seemed reasonable in view of the larger arcs at lower concentrations in
experiments reported here (e.g. Fig.
2 and after dispersion of pheromone in
Fig. 4).
Arousal, duration and direction of the circling response
At high CSSG concentrations, animals responded quickly, within 0.2 s
of encountering CSSG. If their trajectory did not take them back into the
CSSG, their path began to straighten out in less than a second. When a worm is
circling rapidly in response to CSSG and re-enters the CSSG location, it
usually continues circling in the same direction; however, worms can change
their direction of circling, though usually only after its path has begun to
straighten out. With low concentrations of CSSG, passing through the CSSG
usually produced a brief deflection of their path. Accordingly, this behavior
was modeled with a fast rise in arousal of circling, a decrease of circling
arousal beginning within a second, and a 50% chance of changing direction of
circling when the circling rate was less than a given level (in the
illustrated simulations, only at circling rates of <100°
s–1). The curve used to model the arousal and duration of
circling behavior over time is illustrated in
Fig. 5D. The initial direction
of turning for any particular response and the possible change in direction at
low turning angles were determined probabilistically by comparison with random
numbers, with responses occurring equally often to the left as to the right.
Since worms also spontaneously turn, although never at such rapid rates as
when stimulated with pheromone, the model also includes spontaneous slow turns
in either direction in the absence of pheromone.
Dimensions and concentration of the pheromone trail
An estimate of the concentration of CSSG expected to be in a trail is based
on data and anecdotal information in previous publications, as follows.
Average excretion rates of CSSG by female N. succinea ranged from
20 µg h–1 to 150 µg h–1 with an
average of 68.5±12.5 µg h–1
(Hardege et al., 2004).
Swim speeds of females have not been quantitatively reported; however,
female N. succinea have been reported to swim slower than the
non-aroused males. For example, Townsend
(Townsend, 1939a) comments
"The mechanics of the spawning response appears to be entirely similar
in the two sexes but may be more readily observed in the female because of the
slower swimming." Lillie and Just's original publication on spawning in
these animals (Lillie and Just,
1913) says: "males...dart rapidly through the water...The
much larger females then begin to appear, usually swimming laboriously through
the water." We have made similar observations (J.L.R., unpublished
data). Since the non-aroused males swim an average speed of 50 mm
s–1 (the parameter used in the simulation model), for
purposes of estimating a `typical' pheromone concentration, we use here an
estimated female swim speed of 25 mm s–1, i.e. half that of
the non-aroused males.
Swarming females typically range from 3 to 8 mm in diameter (a
representative female, with a diameter of 6 mm, is shown in supplementary
material Fig. S1). If a female leaves a cylindrical trail of approximately the
same diameter, the concentration of CSSG ([CSSG]) in the trail can be
calculated as follows:
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Thus for a `typical' female, [CSSG] in a trail 6 mm in diameter, excreted at a rate of 70 µg h–1 while swimming at 25 mm s–1, would be in the range of 10–9 to 10–8 mol l–1. Owing to uncertainties in this calculation, the model includes user-adjustable variables for pheromone concentration, trail width, and dispersion of the pheromone in response to simulated worms passing through it.
Simulation results
General observations of the simulated behavior
The movement of male worms with and without a simulated pheromone trail
present was simulated. As described in Materials and methods, the speed and
direction of swimming are calculated at each interval, dt, based on
whether simulated pheromone is present at the location of the worm and their
associated concentration–response and arousal curves
(Fig. 5). Without pheromone
present, the simulated worm made occasional small angle turns
(Fig. 6A) but rarely turned
where the trail would be simulated when pheromone is present.
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Higher concentrations of simulated CSSG yielded complex circling behavior, comparable to behaviors observed with real worms encountering higher concentrations of pheromone: multiple turns, dispersion of the pheromone trail, many encounters with the trail as the simulated worm circles back, etc. Examples of complex circling responses in response to log[CSSG]=–8.0 and –7.5 are illustrated in Fig. 6E and F, respectively. In performing these multiple circles, the worm can `explore' a considerable length of trail even though it may not `track' along the pheromone trail for as long a distance at each encounter as occurs with lower concentrations of pheromone. In Fig. 6F, although the pheromone becomes dispersed further from the trail than in Fig. 6E, the animal's actual swim trajectory, represented by the thin black line, generally consists of smaller, faster circles than in Fig. 6E, but the higher concentration of pheromone must be spread more broadly before the simulated animal can `escape' from the cloud of pheromone.
After observing the general properties of the behavior in response to simulated pheromone trails, the simulated behavior was examined quantitatively to determine whether pheromone responses led to significant increases in encountering the trails, following the trails, and finding females on the trails.
Multiple encounters with the pheromone trail
To compare the number of times that the simulated worm crossed the
pheromone trail with and without pheromone present, the behavior of the male
worm was simulated for [CSSG]=0 (i.e. no trail width), and
log[CSSG]=–8.5, –8.0 and –7.5. For each test, the male worm
was launched at an angle of 30° from a distance of 5 cm (perpendicular
distance) from the pheromone trail. In ten trials at each concentration the
number of trail crossings were: no pheromone, 1 (no variation); –8.5,
1.7±0.4; –8.0, 14.3±2.8; and –7.5, 42.5±6.4
(P<0.001, ANOVA on ranks).
Trail-following and length of trail encountered
At each concentration, the longest distance that the simulated worm
traveled along the trail before veering off in another direction was measured.
The maximum length of simulated trail-following observed in all simulation
experiments was 37 cm. In ten trials, the average maximum tracking
lengths were: no pheromone (taken as the width of dispersion from a single
crossing, if a trail had been present), 2 cm; –8.5, 10.5±3.6 cm;
–8.0, 4.8±0.3 cm; and –7.5, 5.3±0.4 cm
(P<0.001, ANOVA on ranks).
The relatively shorter maximum tracking lengths at higher pheromone concentrations is due to the fact that the simulated worms usually circled and hence did not `track' along the chemical trail. However, since they often circled back onto the trail many times, they also often encountered a considerable length of the trail overall. Adding up the length of `disturbed pheromone', the total length of trail encountered (out of a total possible length of 75 cm) was: no pheromone (only a single crossing of the trail, equivalent length that would have been dispersed), 2 cm; –8.5, 11.7±3.7 cm; –8.0, 15.9±2.8 cm; and –7.5, 26.9±5.3 cm (P<0.001, ANOVA on ranks).
Probability of encountering a female on the trail
To determine whether the search strategy simulated in this model would
result in a more likely encounter with a female on the trail, simulated males
were `launched' at various angles from a start position of 5 cm (shortest
distance of a perpendicular path) from the CSSG trail and 20 cm to the
left of a virtual female on the trail (see inset of
Fig. 7). The virtual female was
thus at an angle of about 15° from the male's starting position (where
0° is parallel to the pheromone trail towards the right). Simulations
without a pheromone trail present (trail of width `0') placed the simulated
female in the same relative position with respect to the male.
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Overall, considering all 720 swim trajectories, under control conditions (i.e. no pheromone trail present), the simulated male encountered the female only 6% of the time. With pheromone present, the percentage of trials that resulted in encountering the female increased significantly, approximately doubling at log[CSSG]=–8.5 and increasing approximately threefold at log[CSSG]=–7.5 (ANOVA, P<0.001; all concentrations significantly greater than control, P<0.05, Tukey's test; Fig. 7B).
Thus, the model not only mimics many aspects of the worm's real behavior in response to pheromone trails, it also shows that this pheromone-guided behavior can help male worms find their mates.
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Behavioral data in this paper show that linear distributions of pheromone
can stimulate turning and increase swim speed of male worms. This stimulus
geometry differs from all previous studies of N. succinea, which
tested males either with a point stimulus
(Hardege et al., 2004;
Ram and Hardege, 2005;
Ram et al., 1999;
Zeeck et al., 1998b), or with
a uniform `bath' concentration (Ram et
al., 1999; Zeeck et al.,
1998b). By developing the `rounded heart' aquarium configuration,
we were able study encounters of animals with linear trails. Upon encountering
low concentration trails of pheromone at an oblique angle in the present
study, male worms sometimes swam directly along the trail, an average distance
of 8 cm. At higher concentrations of pheromone, albeit still lower than the
amount needed to elicit spawning, circling behavior stimulated by the
pheromone results in even longer sections of the pheromone trail being
explored.
The use of computer simulation enabled us to test hypotheses that would
have been difficult to investigate otherwise. This study used computer
simulation to examine whether the responses of male N. succinea would
facilitate mating encounters of male worms with females. Computer simulation
enabled the repetition of experiments and the testing of a large range of
pheromone concentrations, swimming directions, etc. that would have been
well-nigh impossible to accomplish with the animals themselves because of some
of their biological characteristics. As noted in the Introduction, once males
get close enough to dance with the females, the females `spawn out' and die,
which would prevent a repeat of the experiment with the same animal under the
same or different conditions. Although males can respond to the pheromone
repeatedly, they too weaken after repeated spawning
(Ram and Hardege, 2005).
Moreover, the need for filtering and/or changing of water when working with
pheromones may cause disturbance of animals from transfer or turbulence. The
need for calm weather for the nuptial dances to occur has been noted
anecdotally (Costello and Henley,
1971). Computer simulation is, of course, not affected by the
vagaries of weather and can readily re-run experiments.
Computer simulation of the responses of male worms to pheromone, based on previous observations of female excretion of pheromone and of male turning and acceleration aroused by the pheromone, approximately reproduced the behavioral responses to linear trails of pheromone observed in the present study. By setting parameters using data from prior publications, the model was created without the circular reasoning that would result from choosing parameters from the behavior to be modeled. Quantitative estimates of pheromone sensitivity do, however, differ somewhat among the various previous publications, possibly because they were obtained with animals from different global locations (Woods Hole, MA, USA and Isefjord, Denmark) that may differ in salinity, temperature, early or late season of experiments, etc. The sensitivity and arousal curves used in the present model therefore produce only a representative set of responses. Variations in these parameters can be used in future studies of the robustness of the simulated behavior over a range of sensitivities, and effects of salinity and seasonality on reproductive behavior and the sensitivity to pheromone can be compared to predictions from the model.
The simulation model was further applied to answer the question of whether this behavior would be likely to increase the likelihood that a male would encounter a female. For example, there is no guarantee upon following a trail that the male would be heading in the right direction. Would heading in the right direction along the trail only about half of the time still result in more frequent encounters with females than ignoring the pheromone trail altogether? Application of the simulation model to this question demonstrated that simulated male worms do indeed encounter virtual females on the trail significantly more frequently than would occur by chance.
The above studies support the hypothesis that male N. succinea can
find females at the water surface by following pheromone trails excreted by
the females. Previous descriptions of N. succinea mating behavior
have been little more than anecdotal. Lillie and Just
(Lillie and Just, 1913)
commented that "When a female appears she is soon surrounded by several
males... which swim rapidly in narrow circles about her." Townsend
(Townsend, 1939b), commenting
on the lack of detail regarding the spawning behavior of another species, the
Atlantic Palolo worm, by Mayer (Mayer,
1908) says that he "gave no fuller account of the swarming
reactions... than that it swam westward, spiraling in a clockwise
direction." A more recent commentary
(Costello and Henley, 1971) on
N. succinea notes only that "males will appear first....The
females appear later; fewer in number and swimming more slowly than the
males....males which approach within a certain orbit will deviate from their
original spiral paths to swim actively around her in rapidly narrowing
circles..." Quantitative videographic studies of the behavior prior to
spawning would help to determine whether details of the behavior match that
predicted by the model.
A constant release of CSSG by females is assumed in the pheromone trail
hypothesis, but the moment-to-moment pattern of excretion is actually unknown.
Townsend (Townsend, 1939b) and
others (Hardege et al., 2004)
have described the large amounts of pheromone released by females over periods
of hours. For example, Townsend (Townsend,
1939b) measured the accumulated amounts of `spawning inducing
material' excreted into a bowl of seawater by a swimming female in successive
2 h time periods. Although such measurements document the large total amounts
of pheromone released by female worms, measurements of cumulative amounts over
long periods give no details as to whether the release may be pulsatile,
directional or constant. Measurements with a finer time and spatial resolution
are needed. Ultimately, simulations modeling both male and female behavior
would provide the best test of the roles of pheromone trails in reproductive
success.
This paper demonstrates how a pheromone may have a dual use, eliciting one
type of behavior at high concentration and short range, and a different,
though related, behavior at longer distance and lower concentrations. CSSG has
previously been clearly demonstrated to be a male spawning pheromone for
N. succinea at high concentrations, above 10–7 mol
l–1 (Ram et al.,
1999). This paper shows that the `non-spawning' swim arousal
effects of lower concentrations of CSSG can be exploited by females as a
seductive signal to attract and guide male worms to their spawning encounters.
Previously, Painter et al. (Painter et al.,
2003) investigated attraction and mating effects of attractin, a
waterborne peptide pheromone in the marine gastropod Aplysia, and
concluded that they could not distinguish multiple activities of the pheromone
from an alternative interpretation in which the main effect is to increase the
`desire to mate'. In fact, attractin alone does not cause significant
attraction of animals but only facilitates turning in t-maze tests when other
attractive factors (e.g. a potential mate) are present
(Painter et al., 2003). The
present study is the first to show that the pheromone alone is enough to cause
both the spawning response (Ram et al.,
1999) and the behavioral responses that can result in
mate-finding.
Overall, the function of the CSSG pheromone is interesting for future
studies on many levels. Although Ram et al.
(Ram et al., 1999) reported
that electrophysiological responses could be activated by CSSG applied near
the anterior of semi-intact male worms, the sensory receptors for the
pheromone have yet to be clearly identified. The `decision-making' process by
which electrophysiological activity in the male results in left or right turns
and maintains that direction above a certain stimulus level in response to the
pheromone, as described in the simulation model, is unknown. The molecular
receptors and transduction mechanisms for glutathionergic compounds used as
extracellular informational molecules [in the case of CSSG as a pheromone, and
in hydra as an activator of ingestion behavior
(Sakaguchi et al., 1991)] are
also completely unknown. Moreover, in the female, regulatory mechanisms
controlling the synthesis and release of the pheromone are almost completely
unexplored. Thus, the multiple behaviors elicited by CSSG to mediate
reproduction in N. succinea are an excellent area in which new
molecular, neurophysiological, and reproductive mechanisms may be
discovered.
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Acknowledgments |
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Footnotes |
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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  K. Phillips WORMS FOLLOW PHEROMONE J. Exp. Biol., March 1, 2008; 211(5): ii - ii. [Full Text] [PDF]
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