Auto-spermatophore extrusion in male crickets
Department of Biology, Faculty of Science, Okayama University, Tsushima-Naka-3-1-1, Okayama 700-8530, Japan
* Author for correspondence (e-mail: masack{at}cc.okayama-u.ac.jp)
Accepted 1 September 2003
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Summary |
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Key words: male cricket, Gryllus bimaculatus, timer, reproduction, sexual refractoriness, spermatophore, terminal abdominal ganglion, genital motor neurone, DUM neurone
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Introduction |
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Thus, the male cricket has a reproductive cycle consisting of the
pre-copulatory mating stage (MS) and post-copulatory sexually refractory stage
(RS). The mating stage is here defined as the interval from the onset of the
calling song to spermatophore extrusion in copulation. The sexually refractory
stage is the interval from spermatophore extrusion to the onset of the calling
song. This refractory stage is further divided into the two substages: the
first refractory stage (RS1) between spermatophore extrusion and spermatophore
preparation, and the second refractory stage (RS2) between spermatophore
preparation and the onset of the calling song. In Gryllus
bimaculatus, RS1 is normally several minutes if a female is readily
available. However, it is considerably prolonged, occasionally more than 1 h,
if no female is present or the male is subject to stressful conditions
(Nagao and Shimozawa, 1987;
Ootsubo and Sakai, 1992
). In
contrast, RS2 is time-fixed (about 1 h) and is unaffected by the female or by
stress. Thus, it is called the time-fixed sexually refractory stage, and is
presumably under the control of a reproductive timer
(Nagao and Shimozawa, 1987
;
Sakai et al., 1995
;
Ureshi and Sakai, 2001
).
In a previous report, we suggested that the timer is located in the
terminal abdominal ganglion (TAG), because functional inactivation of the TAG
by local cooling lengthened RS2 in proportion to cooling time
(Ureshi and Sakai, 2001). In
order to obtain further insight into the function and localization of the
timer, an electrophysiological approach would be useful
(Truman, 1978
;
Tomioka and Chiba, 1986
).
However, the male does not exhibit the calling song under dissection and
restraint. As long as the male remains silent and unresponsive, there is no
guarantee that the reproductive timer is functioning normally even though one
can obtain some information about time-keeping from the spike activity of
neurones in the TAG.
The male cricket exhibits several types of spermatophore extrusion without
genital coupling, such as pseudo-copulation related spermatophore extrusion,
abortion and self-cycle renewal (Beck,
1974; Sakai et al.,
1991
). Recently, we described a new type of spermatophore
extrusion called auto-spermatophore extrusion, in which a male paired with a
female extrudes the mature spermatophore without any prior courtship or
copulatory actions. Auto-spermatophore extrusion was frequently observed when
males were given stress by cooling of the thorax and/or abdomen after
spermatophore preparation (Ureshi and
Sakai, 2001
). Interestingly, the time interval (SPaSE) from
spermatophore preparation to auto-spermatophore extrusion was comparable to or
slightly longer than RS2 measured by the mating response, which suggested that
auto-spermatophore extrusion occurred shortly after the male finished the
sexually refractory stage. Thus, auto-spermatophore extrusion can be used as
an index of the reproductive switchover, as a substitute for the calling song
or the mating response (Ureshi and Sakai,
2001
).
In the present study, we investigate (i) to what extent, and when, auto-spermatophore extrusion occurs in dissected and restrained males, (ii) whether efferent neurones in the TAG show reproductive stage-specific activity for a period long enough to cover several reproductive cycles, and (iii) whether they have any time-dependent activity during the RS2. The results strengthen our previous conclusion that the reproductive timer is located within the TAG, and demonstrate that the timer functions normally in dissected males even when the TAG is separated from the rest of the central nervous system.
Preliminary results have appeared elsewhere
(Kumashiro and Sakai,
1999).
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Materials and methods |
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Definition of SPaSE
Spermatophore extrusion, which is performed by a strong contraction of the
dorsal pouch, is normally elicited by stimulation of special mechano-sensilla
in the epiphallus of the genitalia (Sakai
et al., 1991). The moment of spermatophore extrusion is indicated
by jerky ejection of the attachment plate of the spermatophore from the dorsal
pouch. In contrast, the onset of auto-spermatophore extrusion is somewhat
difficult to determine. The ejection of the attachment plate of the
spermatophore is performed rather gradually by weak dorsal pouch contractions,
as if it were a discharge of waste. However, the final ejection of the
spermatophore is often accompanied by a relatively strong contraction of the
dorsal pouch and abdomen, with the subgenital plate fully opened. In addition,
it is always followed by the rhythmic movement (1/6 s) of the phallic complex
and abdomen, which is not different from that seen after normal spermatophore
extrusion (Kumashiro and Sakai,
2001a
). Thus, the moment of auto-spermatophore extrusion was
defined as the occurrence of a relatively strong contraction of the dorsal
pouch and abdomen followed by their rhythmic contractions. The time interval
between spermatophore preparation (SP) and auto-spermatophore extrusion (aSE)
is abbreviated as SPaSE (the term `spermatophore preparation' is equivalent to
`spermatophore protrusion', which has been used previously).
Induction of auto-spermatophore extrusion
To measure the rate of occurrence of auto-spermatophore extrusion and
variation in SPaSE times, experiments were conducted under four different
conditions. If males did not show auto-spermatophore extrusion within 2 h
after spermatophore preparation, observations were terminated.
In (2-4), males were dissected as soon as they had finished spermatophore preparation. The male was allowed to contact a restrained female with his antennae all the time.
Irritating stimulation of the genitalia
Following spermatophore preparation, males were restrained as in the second
experimental condition (Restrained). Two stimulation methods were used to
irritate the genitalia with foreign objects or by injury
(Sakai et al., 1991). For
stimulation with foreign objects, a piece of plasticine was pushed into the
genital cavity and petroleum jelly was smeared on the cerci, pouch, epiphallus
and ventral lobes. For irritation by injury, the ventral lobes were cut
horizontally with scissors. Bled hemolymph coagulated around the phallic
complex and served as an irritating stimulus. After either treatment, the male
was paired with a female in a beaker and any induced auto-spermatophore
extrusion was noted.
Abdomen-opened and restrained males often discharged diarrheatic feces
during the operation. In the regular experiments feces were removed with a
string of paper as soon as possible because they may cause an early induction
of auto-spermatophore extrusion through persistent irritation of the genitalia
(see Sakai et al., 1991).
However, such feces were intentionally left in some experiments to see if they
could induce auto-spermatophore extrusion soon after their feces discharge in
RS2.
Spike recording
The above procedures allowed quantitative determination of the occurrence
and time of auto-spermatophore extrusion but did not allow spike recording
immediately after spermatophore preparation because of the time required for
making preparations. Thus, in electrophysiological experiments, the operation
and recordings were performed before the induction of spermatophore
preparation. This procedure was somewhat inefficient because the success rate
in inducing spermatophore preparation was as low as 50%.
The treatment for recording has been reported previously
(Kumashiro and Sakai, 2001b).
The male was fixed on a cork plate and the abdomen opened dorsally. The
intestines, accessory glands, testes, periproct and cerci were removed.
Particular care was taken not to stimulate the phallic complex mechanically
because it caused a rapid induction of spermatophore extrusion. Nerve roots
emanating from the TAG were left intact except for 10v (cercal motor nerve)
and 10d (cercal sensory nerve). In most experiments, one dorsal pouch nerve, a
branch of the genital nerve separated from 10v, was cut for recording and the
other was left intact. In some experiments, the dorsal pouch nerves were cut
bilaterally to record spikes on both sides. To isolate the TAG from the rest
of the central nervous system, the connectives were cut between the 6th
abdominal ganglion and the TAG.
Extracellular spikes of the dorsal pouch motor neurone (mDP) were recorded from the cut end of the dorsal pouch nerve with a glass suction electrode, amplifier (AVM-11 Nihon Kohden, Tokyo, Japan) and data recorder (PC204A Sony, Tokyo, Japan). Data were analyzed with a thermal array recorder (RTA-1100 Nihon Kohden). Spike histograms were made every 1 min with a personal computer.
Artificial elicitation of spermatophore extrusion and induction of
spermatophore preparation during spike recording
Normal spermatophore extrusion in dissected and restrained males was
elicited by stimulation of cavity hairs
(Sakai et al., 1991) in the
genital cavity with a model of the copulatory papilla of the female.
Spermatophore preparation was induced by placing the male's antennae onto the
female body, which was restrained in front of the male during recording
(Kumashiro and Sakai,
2001b
).
Backfilling
To stain dorsal unpaired median (DUM) neurones innervating genital organs
including the dorsal pouch and guiding rod, backfilling was carried out with
NiCl2 (1 mol l-1) through the cut end of the genital
nerve distal to the nerve branch innervating the accessory glands. After 24 h
incubation at 4°C, the TAG was cut out, reacted with rubeanic acid, fixed
with formaldehyde (Sakai and Yamaguchi,
1983) and intensified by silver impregnation methods
(Bacon and Altman, 1977
).
Statistical analysis
For statistical analysis, the Mann-Whitney U test was used, and
the significance value was set at P=0.05. All estimates of central
tendency are given as median values (M) with 95% confidence intervals
(CI).
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Results |
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Following this, the abdomen tip slowly moves up and down with high tension (Fig. 1Biii,iv). The extruded spermatophore is gradually pushed backward and is finally rubbed off (Fig. 1Bv). The abdomen and genitalia then began a rhythmical movement with a periodicity of approximately 6 s (Fig. 1C). Auto-spermatophore extrusion in dissected males was essentially similar to that of free-moving males.
Auto-spermatophore extrusion rate and SPaSE in different
conditions
The occurrence of auto-spermatophore extrusion and the time interval
(SPaSE) between spermatophore preparation (SP) and auto-spermatohore extrusion
(aSE) were analyzed in males exhibiting auto-spermatophore extrusion within
the 2 h observation period under the four different experimental
conditions.
(1) Auto-spermatophore extrusion in the free-moving condition was examined in males paired with a female. Almost all (98.3%) of the males (N=414) showed normal spermatophore extrusion during copulation with females within the 2 h observation period. Only seven (1.7%) showed auto-spermatophore extrusion without prior courtship or copulatory attempts (Fig. 2A, above). The SpaSE periods were 55.5-69.5 min (median 63.2 min, CI 62-64.7, N=7; see small upward deflections between 50-70 min), and were comparable to an RS2 of 68.5 min (CI 54.7-80.3, N=414; Fig. 2A, below) measured by the calling song. (2) When males (N=65) were restrained on the cork plate to cause stress, the rate of auto-spermatophore extrusion increased to 30.4%. Males that exhibited auto-spermatophore extrusion under the restrained condition had a SPaSE of 92.8 min (CI 69-104, N=17; Fig. 2B, above), which was significantly longer than 63.8 min (CI 56.2-70.8, N=56; Fig. 2B, below) for RS2 measured by the calling song. The remaining 39 males (69.6%) did not show auto-spermatophore extrusion during the 2 h observation period. When they were paired with females after the end of observations, 12 males exhibited auto-spermatophore extrusion within 10 min and 27 performed courtship. (3) In addition to restraint, when males (N=23) were partially dissected, auto-spermatophore extrusion increased up to 78.3%. These males had a SPaSE of 67.5 min (CI 58-74.2, N=18; Fig. 2C, above), which was not significantly different from 63.5 min (CI 61.2-67.5, N=23; Fig. 2C, below) for RS2 measured by the calling song. (4) Males (N=42) with the abdomen fully dissected showed the highest rate of auto-spermatophore extrusion (80.9%). The SPaSE was 69.8 min (CI 63.7-87.5, N=34; Fig. 2D, above), which was significantly longer than 63.6 min (58.5-66.8, N=42; Fig. 2D, below) for RS2 measured by the calling song.
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Auto-spermatophore extrusion in connective-cut males
Auto-spermatophore extrusion was examined in males with the terminal
abdominal ganglion (TAG) separated from the rest of the central nervous
system. All the males showed auto-spermatophore extrusion. The SPaSE was 61
min (CI 50.2-73.5, N=31; Fig.
3A, above), which was not significantly different from 65 min (CI
57.2-71.2, N=31; Fig.
3A, below) for RS2.
In the experiments reported so far, the operation always started soon after spermatophore preparation and finished within 10 min. To check the possibility that auto-spermatophore extrusion in dissected males might be caused by the delayed effect of operative shock, both the dissection and the connective cut were made at six different times (0-10, 20-30, 30-40, 40-50, 50-60, 60-70 min) after spermatophore preparation. The SPaSE values obtained from six such groups was 68.2 min (CI 51-85.5, N=30) compared with 65.8 min (CI 57-71.2, N=30) for RS2 measured by the calling song. There was no significant difference between the SPaSE and RS2 values. For comparison, the same operation was performed in males in the mating stage. They showed auto-spermatophore extrusion only 2.8 min (CI 2.0-6.8, N=27) after the operation.
These results indicate that the SPaSE in TAG-separated males is comparable to RS2 measured by the calling song, and that auto-spermatophore extrusion in dissected or/and connective-cut males was not due to operative shock or to the connective cut.
Effects of irritating stimulation of the genitalia on the induction
of auto-spermatophore extrusion
The genitalia of intact males (N=17) were irritated with
plasticine and petroleum jelly, and the males then tested in the free-moving
condition. Three (17.6%) of them showed auto-spermatophore extrusion
(SPaSEs=20.2, 49.2 and 66.5 min). The remaining 14 (82.4%) did not show
auto-spermatophore extrusion but recommenced courtship later with an interval
of 62.2 min (CI 56.3-72.7, N=14). On the other hand, in males with
the ventral lobes excised (N=6), five showed auto-spermatophore
extrusion. Their SPaSE values (14.8, 22, 30, 32.3, 59.3 min) were much shorter
than their RS2 values, except for the last one.
A similar experiment was performed using TAG-separated males in which the genitalia were stimulated with diarrheatic feces discharged during the operation. All the males showed auto-spermatophore extrusion, with a SPaSE of 41.7 min (CI 35.3-50.7, N=21; Fig. 3B, above) which was significantly shorter than both 67.5 min (CI 59.5-75.2, N=21; Fig. 3B, below) for the RS2 measured by the calling song and 61 min for the SPaSE in TAG-separated males with no feces (Fig. 3A, above).
These results indicate that adverse stimulation of the genitalia after spermatophore preparation induces SPaSE shortening, particularly in TAG-separated males.
Motor neuronal response to genital stimulation and its spontaneous
activity after spermatophore extrusion
Spike recording from the dorsal pouch nerve indicated that mDP started
bursting after a relatively long latency following genital stimulation by the
model of the female copulatory papilla. In the recording shown in
Fig. 4A, mDP did not respond
for the first 4.3 s after the onset of stimulation. Then, the discharge
increased up to 120 Hz but abruptly stopped after 0.6 s. It gradually
recovered over several seconds during the spermatophore transfer phase
(horizontal bar in Fig. 4A).
This spiking profile occurred when stimulation was removed immediately after
the start of bursting (see legend of Fig.
4A), which indicates that it is centrally generated and lacks a
sensory component.
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After the spermatophore transfer phase, the activity of mDP changed into rhythmic bursting (1/6 s) at an intraburst frequency of 95 Hz (Fig. 4A). The burst was in synchrony with movement of the phallic complex and abdomen. If the male's antennae contacted a female, the rhythmic bursting gradually accelerated and finally stopped before spermatophore preparation. However, if female stimulation was not given, some males did not exhibit spermatophore preparation for more than 1 h. An examination of mDP spike activity (Fig. 4B) showed that the rhythmic bursting also continued for more than 1 h, though the spike rate slightly decreased around 30 min. This pattern was observed in all the preparations tested (N=20). A similar pattern of spike discharges was also seen in males with the connectives cut. These results indicate that the rhythmic bursting of mDP following spermatophore extrusion remains unchanged until the occurrence of spermatophore preparation.
mDP and small neurone activity after spermatophore preparation
During continuous recording of mDP for more than 2 h, the reproductive
stage could be switched experimentally 2 or 3 times from the mating stage to
the sexually refractory stage, using the female and artificial spermatophore
extrusion technique (Fig. 5).
During subgenital plate opening (the left end of the white bar in the first
line in Fig. 5A), 45 s prior to
spermatophore preparation, mDP became silent. At 23 minafter spermatophore
preparation, the spontaneous discharge of mDP appeared
(Fig. 5A, asterisks, line 2),
and continued sporadically with no steady increase or decrease. At 65 min, the
genitalia were stimulated again to elicit spermatophore extrusion
(Fig. 5B, line 1). Spike
discharge vigorously increased in a manner similar to the first cycle. The
temporal silence and subsequent spontaneous discharge followed as before. This
time, however, the spontaneous spike rate was slightly higher than before.
About 2 h after the beginning of the recording, the mating stage was switched
into the sexually refractory stage for the third time
(Fig. 5C). mDP showed a similar
pattern as before. The changes in the spike activity of this mDP are shown
quantitatively in Fig. 6A.
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A much smaller spike occurred in the same recording
(Fig. 5). This neurone is
tentatively named x neurone. The discharge pattern was different from any of
the genital motor neurones so far identified
(Kumashiro and Sakai, 2001b).
Its discharge appeared a few minutes after spermatophore extrusion and
disappeared before the occurrence of spermatophore preparation. After that,
the x neurone began to discharge spontaneously at 29 min in the first cycle
(Fig. 5A, line 3, small spikes)
and at 31 min in the second cycle (Fig.
5B, line 3, small spikes). Then, the spike rate gradually
increased and reached a plateau level (Fig.
6B). A quantitative study of x neurone spike size indicated that
it comprised one (59.1%), two (31.8%), three (4.6%) or more than one neurone
(4.6%) in many preparations (N=22). The latency of the first spike
activity appearing after spermatophore preparation in each single x neurone
was 16 min (CI 6-26, N=31) and the spike frequency was 0.48 Hz (CI
0.18-1.18, N=31) when averaged during the 5 min of the tonically
discharging state 30-60 min after spermatophore preparation.
These results indicated that mDP together with the x neurone can be recorded reproducibly for a period of at least several hours to reveal a stage-specific discharge pattern in the male reproductive cycle.
Neurons with such a small amplitude relative to mDP could be dorsal
unpaired median (DUM) neurones, based on similar extracellular profiles
recorded in the locust (Hoyle and Dagan,
1978; Bräunig,
1988
; Kalogianni and
Pflüger, 1992
). To examine this, simultaneous recordings were
made from the left and right dorsal pouch nerve.
Fig. 7A shows the spike
histograms of two x neurones (r/l and l), which were recorded with different
spike sizes. They gradually increased their spike frequencies 20-30 min after
spermatophore preparation. Their actual spikes are shown in
Fig. 7B, in which one of the x
neurones (r/l; inverted triangle) was recorded from both the right and left
dorsal pouch nerves simultaneously while the other (l) was recorded from only
the left dorsal pouch nerve. The spikes of mDPs with large amplitudes in the
right and left records are shown at low gain in
Fig. 7C. There is no crosstalk
between these records, indicating that the simultaneous appearance of small
spikes (r/l in Fig. 7B) is not
due to artifacts. Simultaneous recordings of the same x neurone were obtained
in 10 out of 19 preparations.
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Fig. 8 shows somata of DUM neurones (30-40 µm in diameter) retrogradely stained by backfilling from the distal region of the genital nerve. Ten somata are seen in the three segments (7-9) of the TAG. Based on ten preparations, two in segment 7, four in segment 8, four in segment 9, and two in segment 10 were identified. Together, these results indicate that some of the x neurones are certainly DUM neurones.
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mDP activity associated with auto-spermatophore extrusion
In these experiments, no genital stimulation was used to cause the male
perform auto-spermatophore extrusion. In males with the connectives intact,
mDP, which had stopped discharging prior to spermatophore preparation
(Fig. 9, line 1), began to show
spontaneous discharging sporadically at 33 min after spermatophore preparation
(Fig. 9, line 4). However, it
stopped around 40 min and remained silent. Then, spiking at a high rate
suddenly appeared at 68 min, with a relatively strong burst followed at 69 min
after an irregular burst that was associated with a dorsal pouch contraction
(arrowhead on the bottom line in Fig.
9). In many recordings, the burst rate was 28 Hz (CI 21-74,
N=13), and the intraburst frequency of the subsequent rhythmic burst
was 87 Hz (CI 75-112, N=13). As in
Fig. 5, x neurone spiking also
appeared in the same recording (Fig.
9). Its activation pattern resembled that described previously,
but did not stop discharging after auto-spermatophore extrusion.
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The time of mDP bursting associated with auto-spermatophore extrusion was analyzed in 16 males (Fig. 10A). 60% of the males showed auto-spermatophore extrusion in the 2 h observation period and 40% did not. The interval between spermatophore preparation and the occurrence of a relatively strong burst associated with auto-spermatophore extrusion had a somewhat wider distribution compared with RS2 measured by the calling song. However, the median of the former was 63.7min (CI 54.7-68.3, N=16), which was not significantly different from 65.5 min (CI 64.2-67.3, N=13) for RS2.
To examine the influence of ganglia anterior to the TAG on mDP and x neurone activity, the connectives were cut 10 min after spermatophore preparation (Fig. 11, white arrow, line 2). No change occurred. In this recording, mDP had no spontaneous discharge following spermatophore preparation. At 59 min, the first spike appeared, and a relatively strong burst occurred at 61 min in association with auto-spermatophore extrusion (Fig. 11, line 7). After this, rhythmic bursting followed as in males with the connectives intact (Fig. 9). By contrast, the x neurone began to discharge 24 min after spermatophore preparation and gradually increased its spike rate (not clearly seen in Fig. 11 because of its extremely small amplitude. See inset between lines 5 and 6). Its spontaneous discharge pattern resembled that in the previous records. These results indicated that the activities of mDP and x neurones in TAG-separated males were not essentially different from those in intact males. All the TAG-separated males exhibited auto-spermatophore extrusion. The interval between spermatophore preparation and mDP bursting associated with auto-spermatophore extrusion was 59.3 min (CI 46.8-68.7, N=15), which is not singnificantly different from 66.2 (CI 63.5--69.5, N=13) for RS2 (Fig. 10B).
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Discussion |
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Timer in dissected males
Our experiments indicate that auto-spermatophore extrusion is induced more
frequently under stressful conditions, and that the SPaSE is comparable to
RS2. The SPaSE in males with the TAG separated from the rest of the central
nervous system is also comparable to RS2. Auto-spermatophore extrusion in the
dissected condition was not due to the delayed effect of operative shock
because the SPaSE was not lengthened when the start of the 10 min operation
was delayed. These results strengthen our previous conclusion that the
reproductive timer is located within the TAG
(Ureshi and Sakai, 2001), and
demonstrate that the timer functions normally even in the separated TAG. It is
not clear why the spermatophore is abandoned rapidly after the end of the
sexually refractory stage, as suggested by the fact that SPaSE is not very
different from RS2. If auto-spermatophore extrusion occurs to omit performing
courtship as mentioned above, the rapid switching of the reproductive stage to
the sexually refractory stage by ejecting the spermatophore seems favorable
for males.
Based upon the present results, previous localised cooling experiments and
evidence that the biogenic amine 5-hydroxytryptophan profoundly influences the
reproductive timer when administered locally in the TAG
(Ureshi and Sakai, 2001;
Ureshi et al., 2002
), we
suggest that specialized neurones in the TAG are responsible for time-keeping.
A similar concept of localized dedicated neurones is emerging for another
time-keeping system in insects. The circadian pacemaker appears to be
localized in the lamina-medulla complex of the cricket optic lobes
(Tomioka and Chiba, 1992
) and
in the accessory medulla of the cockroach
(Petri et al., 2002
).
For the role of peripheral input in auto-spermatophore extrusion, it
remains unclear whether sensory afferent input flowing into the TAG is
involved. In the experiments under dissection, all the nerve roots of the TAG
were left intact except the cercal sensory and cercal motor nerves in
TAG-separated males, although the accessory glands, testes and digestive
system were removed. If the TAG was completely isolated, auto-spermatophore
extrusion could have been affected because its occurrence is facilitated by
artificially applied peripheral input, as discussed below. However, the timer
itself in the TAG is considered to function normally because ablation of the
genital organs did not affect RS2, measured by the calling song
(Sakai et al., 1991).
Experiments in vitro preparations are currently underway to examine
this point.
Auto-spermatophore extrusion stimulated by the afference from
genitalia
Irritating stimulation of the genitalia applied artificially causes a
grooming type of spermatophore extrusion in males in the mating stage; males
with genitalia soiled by discharged feces, or with the ventral lobes incised,
show spermatophore extrusion within a few minutes after stimulation
(Sakai et al., 1991). This
behaviour may be appropriate because the spermatophore could have been spoiled
due to problems in the genitalia. The elimination of the spermatophore is not
the result of passive withdrawal by scraping, since all the males subsequently
do not show the recommencement of courtship but show spermatophore
preparation, indicating that the reproductive stage is switched from the
mating stage to the refractory stage via this type of spermatophore
extrusion.
In the present experiments with males in the sexually refractory stage,
stimulation of the phallic complex with plasticine and petroleum jelly had a
minor effect since most of the males did not show auto-spermatophore extrusion
during RS2. In contrast, stimulation of the ventral lobes by injury was much
more effective: the immature spermatophore was ejected not long after the
operation. The male may actively throw the spermatophore away in response to
something abnormal happening in the genitalia. Stimulation of the genitalia
with feces also caused facilitation of auto-spermatophore extrusion in the
TAG-separated males. Their SPaSE was not so short as that in males with the
ventral lobes injured but it was significantly shorter than RS2. This may be
due to the effect of the irritating stimulation together with lack of
inhibition from the ganglia anterior to the TAG, including the brain
(Matsumoto and Sakai,
2000a,b
;
Kumashiro and Sakai,
2001b
).
From these observations, it is concluded that adverse stimulation of the genitalia can facilitate the occurrence of auto-spermatophore extrusion, particularly in males with the connectives transected. This cautions one to be aware of feces discharged during the operation and to remove any as quickly as possible. Otherwise, one may inadvertently mistake the shortened SPaSE for the original preset timer as estimated by RS2.
mDPactivity in the reproductive cycle
So far, long-term extracellular spike recording has been successfully
carried out using simplified preparations of insects. The activity of motor
neurones and brain neurones associated with ecdysis-related motor patterns
were recorded in a developing moth for several hours after dissection
(Truman, 1978). Even longer
recordings, of up to several days, have been made from optic lobe neurones
underlying the circadian rhythm in crickets and cockroaches
(Tyshchenko, 1973
; Tomioka and
Chiba, 1986
,
1992
;
Colwell and Page, 1990
). In the
present experiments, extracellular spike recording from one of the genital
motor neurones was used as a monitor for the reproductive stages and events in
the male cricket. mDP was particularly suitable because it is singly recorded
from the dorsal pouch nerve and its spike burst causes spermatophore extrusion
via deflection of the dorsal pouch
(Kumashiro and Sakai, 2001b
).
The results revealed that mDP shows a characteristic activity during the two
sexually refractory stages: rhythmic bursting in RS1 and sporadic discharge in
RS2. Furthermore, these discharge patterns were recorded reproducibly in the
same preparation for more than 2 h without any change in activity due to
deterioration or artifact.
In the reproductive cycle, in which the stages are switched via
normal spermatophore extrusion and spermatophore preparation, mDP stops
rhythmic bursting when the subgenital plate opens 45 s prior to spermatophore
preparation. Subsequently, mDP begins to discharge spontaneously, but only
sporadically. This makes sense because mDP is a motor neurone responsible for
contracting the dorsal pouch muscles, which are not used during RS2. There is
a weak time-dependency even in the separated TAG: mDP is completely silent in
the initial state following spermatophore preparation and becomes active some
time later. However, the spontaneous spike rate of mDP was too low and
variable among preparations to clarify this time-dependency. More significant
information concerning timer output might be obtained by recording from other
genital motor neurones, such as those innervating the ventral lobes and median
pouch, because they have higher spontaneous frequencies and show gradual
changes in activation patterns after spermatophore preparation
(Kumashiro and Sakai,
2001b).
Nevertheless, the results demonstrate that neurones in the TAG can withstand long-term spike recording in the dissected condition. Conveniently, spike recording is a much more reliable method for monitoring the state of the reproductive stage and the time of events than movement of the phallic complex, because the changes in the spike rate of mDP are so distinct and stage-specific.
mDP activity associated with auto-spermatophore extrusion
In the experiments without genital stimulation, mDP showed
auto-spermatophore extrusion-associated burst activity with a SPaSE comparable
to RS2. However, the burst frequency was much less (28 Hz) than 137 Hz (CI
110-185, N=16) (Kumashiro and
Sakai, 2001b) in normal spermatophore extrusion induced by genital
stimulation (see Fig. 4A).
Furthermore the spike burst in the former was always preceded by irregular
discharges mixed with some weaker bursts, while in the latter strong bursting
occurred only once, with a long latency of several to 10 s after the start of
genital stimulation. This difference was not due to the presence or absence of
a sensory response component in the burst discharge, because mDP neither
responded simply to genital stimulation nor reduced its discharge when the
stimulus was quickly removed at the moment of the bursting
(Fig. 4A). As for stress in
dissected males under recording conditions, no difference was seen between
auto-spermatophore extrusion and normal spermatophore extrusion. There must be
different neural mechanisms underlying the release of auto-spermatophore
extrusion and the trigger of normal spermatophore extrusion that are
presumably intrinsic to the center for spermatophore extrusion in the TAG,
which may include a neural circuit for excitation, inhibition and rebound
excitation to the genital input.
After the spermatophore transfer phase, mDP activity changed into rhythmic
bursting with an intraburst frequency of 87 Hz, which was comparable to 95 Hz
(CI 94-115, N=11) after normal spermatophore extrusion
(Kumashiro and Sakai, 2001b).
The burst cycle was also similar to that (1/6 s) seen after normal
spermatophore extrusion. These changes are reliable indicators of the
occurrence of auto-spermatophore extrusion, even in cases where the
spermatophore is not actually extruded in males using the accessory glands and
testes removed, as in the present experiments. Additionally, it should be
noted that mDP is not a timer neurone and its burst does not indicate the
moment of the time-up itself of the reproductive timer. However, recording mDP
is useful for future studies in even more simplified preparations to monitor
the reproductive state.
Time-dependent neurone activity
The spontaneous spike activity of one or more small neurones (x neurones),
simultaneously recorded with mDP, had a much clearer time-dependency than mDP;
firing began 10-30 min after spermatophore preparation, then gradually
increased in frequency and reached a plateau level. The tiny spike amplitudes
suggest that these are not motor neurons
(Hoyle and Dagan, 1978;
Bräunig, 1988
;
Kalogianni and Pflüger,
1992
). Although all the x neurones were not tested, some showed
simultaneous spike discharge in the right and left dorsal pouch nerves,
indicating that they are dorsal unpaired median (DUM) neurones, as reported in
the thoracic ganglion (Evans and O'Shea,
1977
; Hoyle and Dagan,
1978
; Gras et al.,
1990
; Thompson and Siegler,
1991
; Burrows,
1996
; Baudoux and Burrows,
1998
; Grolleau and Lapied,
2000
; Bräunig and Pflüger, 2001) and the abdominal
ganglion (Lange and Orchard,
1984
) of the locust. Other x neurones, recorded from only one side
of the dorsal pouch nerve, may also be DUM neurones, based upon similar
activation pattern and spike rates. The missing spontaneous contralateral
spike may have been hidden under the background noise because of the small
size. In addition, we stained several DUM neurones, together with genital
motor neurones including mDP, by backfilling through the genital nerve. The
pattern of soma distribution was similar to that reported in the TAG of the
male cockroach (Sinakevitch et al.,
1996
). At present, it is unknown which DUM neurone somata send
their axons to the dorsal pouch nerve, from which we recorded x neurones with
mDP, because backfilling through the thin dorsal pouch nerve hardly stained
DUM neurones. The presence of time-dependent activity in the TAG suggests that
the timer output may not be `all or none', as seen in behavioural output such
as the sudden emission of calling after the 1 h silence, but rather is graded
to influence different neurones at different times after spermatophore
preparation. It is necessary to investigate DUM neurones further and to
determine the origin of their time-dependent activity.
In conclusion, the present results strengthen our previous suggestion that the reproductive timer is located within the TAG, and demonstrate that the timer functions normally in the separated TAG once it is triggered at spermatophore preparation. The gradual increase in the spike activity of efferent neurones in the TAG gives an insight into the fashion of the timer output. The next step is to seek timer neurones in simplified preparations.
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