J. Exp. Biol. (1965), 43, 4S3-435
Printed in Great Britain
423
ON THE INITIATION OF SPERMATOPHORE FORMATION
IN THE AFRICAN MIGRATORY LOCUST, LOCUSTA
MIGRATORIA MIGRATORIOIDES
REICHE AND FAIRMAIRE
BY G. E. GREGORY*
Department of Zoology, University College of South Wales and Monmouthshire,
Cardiff
(Received 30 September 1964)
INTRODUCTION
The spermatophore of the African migratory locust consists of (a) a dilated reservoir
composed of two parts, the first and second bladders, which during copulation
remain within the copulatory organ of the male; and (b) a long tube which passes into
the spermatheca of the female. A detailed account of the mode of formation of the
spermatophore has been given elsewhere (Gregory, 1965). The primary purpose of
the present study was to discover the main mechanisms by which the process of
formation is initiated and controlled, but to do this it was necessary to investigate
also the mechanisms controlling the sexual behaviour of the male prior to spermatophore formation. The study is not concerned with the causes of the sexual drive which
is a prerequisite for the occurrence of sexual behaviour.
MATERIAL AND GENERAL METHODS
Of the locusts used in these experiments the males had been segregated from
females in partitioned cages for a week or more, the males being in one half of a
cage and females in the other half. Such males showed an increased readiness to
exhibit sexual behaviour but the patterns of sexual behaviour they displayed were
identical with those of stock males. In all experiments, the females employed were
normal, mature animals obtained from stock
All surgical operations were done under a binocular dissecting microscope ( x 875)
with the animal under continuous carbon dioxide narcosis. For operations upon the
front of the head the animal was held head uppermost, but for all other operations
the animal was fastened down with loops of plasticine on to an 'operating table'
consisting of a small sheet of glass covered with a layer of plasticine moulded to fit
the animal's body. During operations incisions in the body wall were held open by
means of small hooks of 5 amp. fuse wire anchored to the surface of the operating
table with pieces of plasticine. At the end of every operation wounds were sealed
by fusing together the cut edges of the body wall with a hot needle and covering the
area with paraffin wax; this reduced post-operational blood loss almost to nothing.
• Present address: Department of Insecticides and Fungicides, Rothamsted Experimental Station,
Harpenden, Herts.
27-2
424
G. E. GREGORY
The animal was then numbered with black cellulose paint on the dorsal surface of the
pronotum. Recovery from operations took usually from a half to 2 hr., depending
upon the length of the operation. Mortality, even though operations were not done
under aseptic conditions, was low (less than 10 %). Details of the individual operations
are given in the appropriate parts of the text. All the times quoted refer to a temperature of 33—35° C
SEXUAL BEHAVIOUR IN THE MALE
The sequence of male behaviour culminating in the formation of a spermatophore
can be divided into four clearly denned phases: approach; mounting; copulation;
and spermatophore formation. Detailed accounts of these various phases have been
given elsewhere, approach and mounting having been described by Mika (1959),
who classed them together as 'preparation for copulation' (Paarungseinleitung),
copulation having been described by Boldyrev (1929) and spermatophore formation
by Gregory (1965), so that here only the salient points of the phases will be mentioned.
It should be pointed out that throughout this account the term ' copulation' is used to
mean simply the mechanical union of the male and female genitalia and is not intended
to imply the formation and passage of a spermatophore. For the purposes of behavioural analysis copulation and spermatophore formation, though largely overlapping
in timing, are treated as distinct phases of behaviour.
The behaviour sequence begins when the male first perceives the female, whereupon he orientates to face in her direction and then starts to move towards her with
a series of characteristic, short, jerky steps. During this approach the male usually
pauses once or twice and makes rapid vibratory movements of the maxillary and
labial palpi and side-to-side ' peering' movements of the anterior end of the body.
The latter are probably connected with the estimation of the distance away of the
female, for they closely resemble the movements, also termed ' peering' movements,
studied in the desert locust, Schistocerca gregaria, by Wallace (1958, 1959).
When near enough the male either climbs, or more frequently jumps, on to the
back of the female, this marking the beginning of the mounting phase. The male
clings to the female by means of his first two pairs of legs, the third pair playing no
part in attachment. While he is mounted upon the back of the female, the male
occasionally makes side-to-side movements of his head, with his palpi closely applied
to the female pronotum; the significance of these is unknown.
Copulation normally follows soon after the male has mounted, when he lowers his
abdomen to one or other side of that of the female, protrudes his genitalia so that they
may grasp her subgenital plate and inserts his aedeagus into her spermathecal aperture.
Attachment to the female is assisted by the male cerci, which grip the abdomen of
the female on either side of her subgenital plate.
Spermatophore formation begins usually less than 2 min. after the start of copulation (Gregory, 1965), so that the initiation of formation must occur almost immediately
after copulation has begun. However, the fact that a spermatophore is being formed
cannot be recognized externally until the expansion of the first bladder of the spermatophore reservoir, although usually just before this a small quantity of fluid is exuded
on to the pallium at the base of the aedeagus and may be indicative of spermatophore
formation (see below). The first reservoir bladder becomes visible as a small, whitish
Spermatophore formation in the African migratory locust
425
mm
swelling at the base of the aedeagus after 11-15 min. copulation. Then, 20-25
later, the swelling begins to decrease in size as the spermatophore tube is extruded
from the aedeagus and the contents of the reservoir are passed along the tube into the
female. This last process takes from 5 to 18 hr., and when it has been completed
copulation is brought to an end by the male and female separating.
EXPERIMENTAL ANALYSIS OF MALE SEXUAL BEHAVIOUR
The sequence of male sexual behaviour was investigated from two viewpoints: to
•determine what receptor organs were involved in the various phases of the process;
and to discover by what pathways the stimuli were transmitted from these receptors
to the effector organs, the body musculature, genitalia and accessory glands, involved.
Experiments with these objects in view were carried out as follows.
Inactivation of head receptors
To investigate the importance of head receptors in sexual behaviour the various
head structures bearing the greatest concentration of sensory receptors—the antennae,
palpi and eyes—were inactivated, first singly and then in different combinations:
the compound eyes and ocelli; the maxillary and labial palpi; the maxillary and labial
palpi and the antennae; and the antennae, compound eyes and ocelli. The antennae and
palpi were inactivated by amputation and the compound eyes and ocelli were covered
with two coats of black cellulose paint. After treatment the experimental males,
twenty-seven in number, three being used for each experiment, were placed with
females and their behaviour was observed.
It was found that none of these operations interfered markedly with sexual behaviour,
except in the case of males in which all the eyes, both compound and simple, had
been blackened, for these males no longer mounted the female by jumping but
always crawled on to her.
It appeared from these experiments that either head receptors were not especially
important in the control of sexual behaviour, or the stimuli emanating from the female
were sufficiently varied in character to be still perceptible to the male as long as any
type of head receptor remained functional. To investigate the importance of the
head receptors as a whole ten males were operated upon to inactivate at one and the
same time the eyes, the antennae and the palpi, and the behaviour of the males
towards females was then tested, over a period of several days. Control experiments
were carried out by removing the paint from the eyes of three of the males after a few
days and again testing their behaviour.
Sexual behaviour was found to be hampered by these operations to a considerable
extent, being prevented altogether in six of the males. However, when the eyes of
three of these six males were uncovered normal sexual behaviour returned, showing
that it had been prevented only by the lack of functional head receptors. Sexual
behaviour was not prevented in all of the experimental males, however. In these
cases mounting took place only when a male accidentally stumbled across a female,
so that normal approach behaviour was not shown; but despite this, in two cases,
copulation and spermatophore formation did occur. As these males behaved so
differently from the others they were very carefully examined to verify that the head
426
G. E. GREGORY
receptors had been inactivated properly, and this was indeed found to be so. Thus
sexual behaviour is still sometimes possible even in the absence of antennae, palpi
and functional eyes.
The only other head organs bearing receptors which might be of use in sexual
behaviour appeared to be the mouthparts, and so these were inactivated next. At first
this was done by severing their nervous connexions with the suboesophageal ganglion,
but this was difficult to do efficiently and it was found much more convenient simply
to amputate the labrum, maxillae and labium, and to cauterize the hypopharynx
when sealing the wounds. The mandibles were left intact, for it seemed unlikely that
they could carry any important receptors and they formed a good support for the wax
used to cover the wounds. This treatment of the mouthparts was found not to affect
markedly the ability to copulate of otherwise normal males, which thus formed the
controls, and experiments were therefore carried out to determine whether it would
affect the behaviour of males already deprived of antennae, palpi and functional eyes.
Out of the ten males experimented upon only four.showed any sign at all of sexual
behaviour and none of these made any attempt to copulate. None of the other six
males, even when placed upon the backs of females, showed the slightest indication
of sexual behaviour. Thus, apparently, removal of the mouthparts does reduce the
ability of males already deprived of antennae, palpi and eyes to show sexual responses.
From this it appears that the receptors of mouthparts in addition to the palpi can be
of use in recognizing the female. However, the fact that sexual behaviour was not
prevented in all males by inactivation of the eyes, antennae and mouthparts suggests
that there may be other, less obvious head receptors which can be used in recognizing
the female, or that this may be carried out, at least in part, by receptors on the thorax
or abdomen. This latter point is considered further below.
Although head receptors were thus shown to be of less importance in the control
of sexual behaviour than preliminary observations had suggested, some kind of head
sensory structure was required for the performance of the full sequence of sexual
activities. To determine the minimum of receptor-bearing head structures necessary
all except one or two of the major sensory structures were inactivated and the effect
upon sexual behaviour was observed; thirty-nine males were used.
It was found that for completely normal sexual behaviour the minimum of receptorbearing head structures required is two eyes; with less than two eyes functional, males
never jumped on to the female during mounting, but always crawled on to her. It
seems that it is the compound eyes that are really important in this connexion, for
whereas when these were intact males usually jumped on to the female, when only
ocelli were functional jumping on to the female occurred only very rarely and even
then may have been purely fortuitous. The importance of the compound eyes lies
presumably in their use in the estimation of the distance to be jumped (Wallace, 1958,
1959). Although fully normal sexual behaviour required the presence of functional
eyes, sexual behaviour that appeared normal in all respects except for the absence of
jumping was still shown so long as there was present one antenna, or the mouthparts,
provided that one of their palpi (labial or maxillary) remained intact, or one compound
eye or ocellus.
Spermatophore formation in the African migratory locust
427
Inactivation of thoracic and abdominal receptors
Although the male thorax possesses no large, obvious sense organs, it has numerous
sensory hairs which would respond to contact with the female (Haskell, 1956). Of
these hairs, the ones on the sternites and tarsi seemed likely to be of particular importance in sexual behaviour, for these parts are, after mounting, closely applied to the
body of the female. The male abdomen bears, in addition to many sensory hairs,
two types of organs, the tympanal organs and the cerci, which might have some
sensory role in sexual behaviour. It seemed possible also that the aedeagus might
possess receptors, but histological studies gave no evidence of this.
The role of these possible receptor structures in sexual behaviour was investigated
by inactivating each type separately. The thoracic sternites were cauterized and covered
with paraffin wax, the tarsi were treated similarly, their claws being left uncovered
so that they could still grip the female, and the tympani were punctured with a hot
needle. The cerci were inactivated by cautery, by covering them with wax, or by
amputation. Any receptors on the intromittent part of the aedeagus were inactivated
by cautery or by amputation of the tip. After each of these operations the behaviour
of the males was tested with females. Twenty-seven males were used, three for each
test.
It was found that only the cerci appeared to be of particular importance in sexual
behaviour, for inactivation of the other structures produced no visible effect upon it.
However, after any interference with the cerci, even if with only one of them, males
seemed quite unable to copulate, despite their vigorous and sustained attempts,
although approach and mounting behaviour appeared normal. This agrees with the
observation made by Mika (1959). That this result was not due to any other cause is
shown by the fact that when the wax was removed from the cerci of those males to
which it had been applied they copulated and formed spermatophores without
difficulty.
These results showed that the cerci were necessary for copulation but did not
reveal whether they were necessary for spermatophore formation. To investigate this
the cerci were removed from males at various times after copulation had begun and
the effects upon spermatophore formation were studied by observing whether the
first bladder of the spermatophore reservoir became expanded and by dissection
after 1 \-z hr. to check the final state of the spermatophore.
Amputation of the cerci during the early stages of spermatophore formation,
before the expansion of the first reservoir bladder, resulted, in all of the four males
employed, in the immediate withdrawal of the genitalia, bringing copulation to a
premature end. Spermatophore formation was also brought to an end, so that in
none of the males was the first reservoir bladder expanded. This suggests that for
the early stages of spermatophore formation copulation must continue, and for this
the male cerci must be functional. Amputation of the cerci immediately after the
expansion of the first reservoir bladder resulted in only one out of the three males
tested withdrawing his genitalia, and in all three animals spermatophore formation
continued. The spermatophore became fully formed in the two cases in which copulation continued, but in the third male, in the absence of a female, formation could go
only as far as the beginning of extrusion of the spermatophore tube from the aedeagus.
428
G. E. GREGORY
These results show that the cerci are definitely not necessary for the continuation of
spermatophore formation once the first reservoir bladder has become expanded. They
also suggest that, after this point, spermatophore formation can continue independently
of copulation. This is considered further below.
Inactivation of cerebral ganglia
In order to investigate the part played in the control of sexual behaviour by the
cerebral ganglia, composing the brain, these ganglia were inactivated either by
extirpation or by section of the circum-oesophageal connectives joining them to the
remainder of the central nervous system. Both these operations were carried out
through a small, square hole cut in the frons, which was afterwards sealed with wax.
Control experiments were carried out by severing, at one and the same time, the
optic, ocellar and antennary nerves but not the circum-oesophageal connectives.
After recovery each male was placed with a number of females and its behaviour was
observed over a period of several days.
No sign at all of sexual behaviour was shown by any of the six experimental males,
even when they were placed on the backs of females, and none of them survived for
more than 4 days from the time of the operation. The control males, on the other
hand, recovered rapidly and within a few hours exhibited sexual behaviour, normal
except for the absence of jumping in the mounting phase. Both in controls and
experimental animals the sensory input necessary for the initiation of sexual behaviour
was in part maintained (from the receptors of the mouthparts to the suboesophageal
ganglion); despite this, sexual behaviour was prevented when the cerebral ganglia
were inactivated (in the experimental animal) although it continued when the
cerebral ganglia remained functional (in the controls). Thus it would seem that
the cerebral ganglia are necessary at least for the initiation of sexual behaviour.
It was not possible to determine experimentally the importance of the suboesophageal ganglion in the control of sexual behaviour, for this ganglion could not be
inactivated without severing the brain and the head receptors, already shown to be
necessary for sexual behaviour, from the rest of the central nervous system. Thus
whether the suboesophageal ganglion exerts any inhibitory effect, as it does in
mantids (Roeder, 1935), remains to be discovered.
To determine whether the brain and suboesophageal ganglion were necessary for
the later phases of sexual behaviour ten males were placed with females; and at various
times after sexual behaviour had begun the cerebral and suboesophageal ganglia of
the males were inactivated by decapitating the animals, the effects upon copulation
and spermatophore formation being observed.
It was found that copulation was prevented by decapitation of the male after
mounting but before copulation had begun, and brought to a premature end by
decapitation after copulation had begun but before the expansion of the first reservoir
bladder of the spermatophore, the male genitalia being withdrawn and spermatophore
formation ceasing. Only after the expansion of the first reservoir bladder of the
spermatophore did decapitation of the male have no inhibitory effect, and the male
and female genitalia then always remained firmly interlocked and spermatophore
formation always went to completion.
It is clear from these results that once the first reservoir bladder of the spermato-
Spermatophore formation in the African migratory locust
429
phore has become expanded the brain and suboesophageal ganglion are no longer
necessary for the continuation of spermatophore formation. However, it cannot be
concluded positively that these ganglia are essential either for copulation or for the
earlier stages of spermatophore formation, for the termination of these phases of
behaviour may perhaps have been brought about by operational shock rather than
by the inactivation of the ganglia.
Section of the ventral nerve cord-
To investigate the role of the remainder of the central nervous system in the control
of sexual behaviour, both connectives of the ventral nerve cord, which includes eight
ganglia, here numbered I to VIII, were transected, through a ventral incision in the
body wall, in various positions: between ganglia I and II, or II and III, in the thorax;
between ganglia III and IV, at the junction of thorax and abdomen; or between
ganglia VII and VIII, in the abdomen. The nerves passing posteriorly from ganglion
VIII to the genitalia were also transected. Three males were used for each experiment.
Control experiments were carried out by cutting the body wall in the same position
and exposing the nerve cord without cutting it. After recovery the males were
placed with females and their behaviour was observed. At the close of each experiment
the male was dissected to make certain that the nerve cord had been severed completely in the desired position.
It was found that section of the ventral nerve cord between ganglia I and II prevented all sexual behaviour, whereas section in the other positions, although preventing
copulation and spermatophore formation, did not interfere with approach and
mounting. The only exception to this was section of the cord between ganglia II and
III, which affected the proper functioning of the posterior pair of legs and thus
prevented the male from jumping on to the female during mounting, though it did
not prevent mounting from taking place. No movements of the genitalia were seen
in any of the males experimented upon and the bending down of the abdomen alongside
that of the female, which precedes copulation, was prevented by section of the nerves
in all positions except between ganglia VII and VIII and behind ganglion VIII. All
the control males showed completely normal sexual behaviour.
From these results it is evident that for approach and mounting to occur both
ganglion I and ganglion II must be connected to the cephalic ganglia, and for the
male to be able to jump on to the female during mounting ganglion III must be
connected to the anterior as well. Again, for movements of the abdomen, leading to
copulation, to take place some, at least, of the first four abdominal ganglia must be
connected to the anterior ganglia. For copulation itself, ganglion VIII and the genitalia must also be in communication with the anterior parts of the nerve cord.
These experiments, although providing information on the nervous control of
approach, mounting and copulatory behaviour, threw no light on the control of
spermatophore formation, for none of the experimental males was able to copulate
and copulation has to begin before a spermatophore can be formed. To study the
role of the ventral nerve cord in the control of spermatophore formation the cord
was transected at various times after copulation had begun and the effect upon
spermatophore formation was observed. The cord was transected immediately in
front of ganglion VIII, for, as this ganglion is the only one supplying nerves to the
430
G. E. GREGORY
accessory glands (which secrete the spermatophore), it seemed most likely that only
this ganglion would be directly concerned in the control of spermatophore formation.
The method used involved the insertion of a small U-shaped loop of 5 amp. fuse wire
around the nerve cord between ganglia VII and VIII so that its free ends protruded
posteriorly through the ventral body wall and could be fastened to the surface of the
cuticle with wax. The male was then allowed to copulate; after the required interval
had elapsed the wire loop was carefully drawn out from the abdomen and the nerve
cord was cut with fine scissors. The male and female were then kept under observation
for 2-3 hr. to see whether spermatophore formation continued. They were finally
dissected to check the state of the spermatophore and to make certain that the nerve
cord had been severed completely in the desired position. Control experiments were
carried out by inserting the wire loop as described and allowing copulation to take
place without the loop being removed; in these cases spermatophore formation always
went to completion.
In all of the four animals experimented upon it was found that severing the nerve
cord before the first reservoir bladder of the spermatophore had become expanded
stopped spermatophore formation completely, so that expansion of the bladder never
took place. This was so even though, in three cases, copulation continued uninterrupted
for as long as z\ hr. Thus spermatophore formation, certainly in its earlier stages,
requires not merely the continuation of copulation but also control by one or more
of the ganglia anterior to ganglion VIII. Transection of the nerve cord after the
expansion of the first reservoir bladder, however, did not prevent spermatophore
formation from going to completion in any of the three animals tested, showing that
for the later stages of formation—secretion of the spermatophore tube and its extrusion
into the female—control by the anterior ganglia is no longer required.
It appeared that these later stages of spermatophore formation were controlled
solely by ganglion VIII. To verify this males were allowed to copulate and, immediately expansion of the first reservoir bladder of the spermatophore had taken
place, were separated from the females, it being known (see below) that once the
first reservoir bladder had become expanded the presence of the female was no longer
required for spermatophore formation to continue in the male. The last abdominal
ganglion of each male was then extirpated through a ventral incision in the body wall.
Controls were provided by opening the abdomen in the same way, but without
disturbing ganglion VIII. No anaesthetic was used for these operations for it was
found that carbon dioxide narcosis itself arrested spermatophore formation. After
treatment each male was examined periodically to see whether spermatophore
formation continued, as shown by the extrusion of the spermatophore tube from the
aedeagus, and then after \\-z hr. was dissected to check the state of the spermatophore and to verify that operations had been properly performed.
In every one of the five males tested extirpation of ganglion VIII was found to
have stopped spermatophore formation completely, so that even after more than
\\ hr. there was still no indication of the spermatophore tube being formed. In the
control animals the tube was formed and extruded in the normal time. Thus the
stages of spermatophore formation following the expansion of the first reservoir
bladder must be controlled by ganglion VIII alone.
The foregoing experiments showed that for copulation and the earlier stages of
Spermatophore formation in the African migratory locust
431
spermatophore formation to. take place the abdominal ganglia had to be in nervous
communication with those anterior to them. To discover how much communication
was necessary only one of the two connectives of the ventral nerve cord was cut,
either between ganglia III and IV or between ganglia VII and VIII, and the effects
upon sexual behaviour were observed. Previous experiments had already shown that
incisions in the abdominal wall made no difference to sexual behaviour so that no new
controls were required.
It was found that in none of the six males operated upon was copulation or spermatophore formation interfered with. Thus for the bending down of the abdomen
alongside that of the female ganglia IV-VII need be connected to the anterior ganglia
on one side only, and likewise for copulation and spermatophore formation ganglion
VIII needs to be connected to the anterior ganglia on one side only.
This raised the question of whether, when only one side of the ventral nerve cord
was intact, the spermatophore was secreted by the accessory glands of that side only,
or of both sides. Could stimuli passing to ganglion VIII along the connective of one
side activate the whole ganglion or only the one side of it? To answer this question
six males were operated upon as follows; one side of the ventral nerve cord was cut
between ganglia VII and VIII, and the accessory glands were removed, in three of
the males on the cut side and in the other three males on the intact side. The glands
were removed, through a lateral incision in the abdominal wall, by cutting them off
close to the ejaculatory duct, the cut ends remaining being sealed by cautery. The
two groups of males were then compared for ability to form spermatophores. Controls
were provided by removing the accessory glands of one side without cutting either
side of the ventral nerve cord. At the end of the experiments all males were dissected
to check that the operations had been carried out correctly.
It was found that normal spermatophore formation still took place in both groups
of experimental animals as well as in the controls. Thus a normal spermatophore can
be produced even when only one of the two accessory gland masses is present, and
for spermatophore formation it is immaterial whether the remaining accessory gland
mass and the intact connective of the ventral nerve cord are on the same or on opposite
sides of the body. Therefore stimuli arriving at ganglion VIII along a connective of
one side only are able to activate the whole ganglion, which can then initiate secretion
by the accessory glands of both sides of the body.
The role of the copulatory fluid
During copulation a viscous, colourless fluid, here termed the copulatory fluid,
always appeared, usually just before the expansion of the first reservoir bladder of
the spermatophore, at the junction of the male and female genitalia, forming a thin
film over them. Separation of copulating individuals at this time frequently revealed
a small amount of similar fluid filling the distal end of the aedeagal canal. The fluid
was presumed to come either from the female spermatheca, which is known to secrete
a fluid (Gregory, 1965), or from the male genital ducts or accessory glands. Experiments involving the extirpation of these organs pointed to the fluid being secreted by
the male accessory glands.
The production of this fluid during spermatophore formation is interesting in that,
though it may have merely a lubricatory function, serving to assist the extrusion of
432
G. E. GREGORY
the spermatophore tube along the canal of the aedeagus, there seems at least a
possibility that it may have some more complex, chemical role. It may perhaps
stimulate chemoreceptors on and around the genitalia and so have an effect upon the
behaviour of the female and possibly of the male as well.
THE ROLE OF THE FEMALE IN SPERMATOPHORE FORMATION
The presence of the female is essential for the initiation of spermatophore formation,
for this cannot take place until copulation has begun. The results of experiments
already quoted have shown that the presence of the female and the continuation of
copulation are essential also for the subsequent stages of spermatophore formation,
at least up to the expansion of the first reservoir bladder. However, one observation
had suggested that after this point formation of the spermatophore could continue,
certainly up to the stage at which it is ready to be extruded into the female, even though
copulation had come to an end and the female was no longer present. This was verified
by allowing six males to copulate for known, different, lengths of time and then
carefully pulling the females away from them, the effect of this upon spermatophore
formation being studied by observation of the males over the next 30-40 min.,
followed by their dissection to determine the final state of the spermatophore.
It was found, as suspected, that, while removal of the female before the first reservoir
bladder had become expanded always brought spermatophore formation to a halt,
removal of the female after the expansion of the bladder, even if it had only just
occurred, had no such effect. Thus of the stages of spermatophore formation taking
place in the male only those up to and including the expansion of the first reservoir
bladder require the presence of the female.
However, unless the female is present and copulation continues the tube of the
spermatophore is never extruded more than a millimetre or two from the male
aedeagus, for its outer layers seem to dry rapidly when exposed to the air and eversion
ceases, despite the vigorous attempts of the male to continue the process. As a
result of the continuing pressure from the musculature of the male copulatory organ
the spermatophore tube finally bursts. Nevertheless, for the extrusion of the tube
to go to completion only the spermatheca of the female is required (Gregory,
1965). The spermatheca appeared to function here simply as a mould, confining the
spermatophore tube laterally and allowing it to extend only longitudinally. This was
confirmed by inducing males to extrude the tube of the spermatophore along a fine
glass tube, open at both ends and filled with saline, which was fitted over the tip of
the aedeagus just as extrusion of the spermatophore tube was beginning. It was
found that, as long as the glass tube reproduced within fairly narrow limits the
internal dimensions of the average spermatheca, extrusion of the spermatophore
tube would continue, almost to completion, in a perfectly normal manner. Thus,
although copulation with the female is essential for the initiation and continuation
of the earlier stages of spermatophore formation, the role of the female during the
later stages of the process appears to be a completely passive one.
Spermatophore formation in the African migratory locust
433
DISCUSSIONS AND CONCLUSIONS
The results of the present experiments, although not supplying answers to all
of the questions investigated, do make it possible to delineate, in general terras, some
of the mechanisms by which the sequence of male behaviour culminating in spermatophore formation is controlled.
The sequence of male sexual behaviour is initiated apparently by stimuli from the
female being received by the antennae, eyes and mouthparts of the male. This
stimulation presumably leads to activation of a cephalic centre, as occurs in the
cricket Gryllus campestris (Huber, 1953), and this then, by way of ventral nerve cord
ganglia I, II and III, activates the legs, causing approach behaviour to begin. The
importance of the mouthparts, which are particular sites of chemotactile receptors,
strongly suggests that some kind of chemoreception is involved here. Little is known
of what chemical stimuli may come from the female, but an ectohormone, or pheromone (Karlson & Butenandt, 1959), is known to be produced by the male desert locust
(Schistocerca gregaria) (Loher, i960) and is suspected in the male of Locusta (Haskell,
1962). It seems fairly certain that at close range chemotactile activity could be
important both in species recognition and in sex recognition, and that it could also
be a releasing stimulus for the next phase of behaviour.
When the male has moved near enough to the female the pattern of stimuli then
arriving at the cephalic centre brings about the initiation of mounting behaviour. This
also is effected by way of the thoracic ganglia and legs. Both approach and mounting
thus appear to involve the receptors, nervous system and effectors of the head and
thorax only, and this conclusion is further supported by the fact that these phases of
behaviour are entirely unaffected by section of the ventral nerve cord immediately
behind the thorax, whereas such treatment completely prevents the subsequent phases
of behaviour. The cephalic centre controlling approach and mounting probably lies
in the brain, for when this is inactivated no sexual behaviour at all occurs.
The next phase of sexual behaviour, copulation, is controlled through the ganglia
of the abdomen, which activate the muscles of the various segments concerned.
However, it appears that the initiation of copulation is carried out by a centre, or
centres, in front of the abdomen, perhaps in the head, because for copulation to
begin the abdominal ganglia have to be in nervous communication with the ganglia
anterior to them. However, this communication need be maintained, certainly in
the abdomen, on one side of the ventral nerve cord only. This contrasts with the
situation found by Huber (1955) in Gryllus campestris, in which for copulation to
occur the ventral nerve cord had to be intact on both sides.
For copulation to take place in Locusta both cerci are also required. It seems likely
that, as well as acting as clasping organs, the cerci may also have a sensory function
or functions in mating, as they bear numerous sensilla. Haskell (1956) has suggested
that they are mechanically stimulated by copulatory movements and so may influence
the state of excitation of the central nervous system. In Gryllus campestris the cereal
sensilla must be stimulated for full mating behaviour to be shown (Huber, 1953).
The cerci may well also have the function, as has been suggested by Mika (1959), of
guiding the male genitalia into the correct position for copulation.
Of the movements involved in copulation the bending down of the abdomen
434
G. E. GREGORY
alongside that of the female seems to be brought about by the operation of intersegmental mechanisms controlled through some, at least, of the first four abdominal
ganglia (IV-VII). Thus though bending is prevented by section of the nerve cord
immediately in front of ganglion IV, it is not affected by section immediately behind
ganglion VII. After section in this position none of the receptors on the genitalia or
cerci can be used to control bending, and so this must be monitored by receptors on
or in the abdominal segments which stop the bending when the right position of
contact with the abdomen of the female is reached. Protrusion of the genitalia is
controlled through ganglion VIII, the only ganglion supplying nerves to the genitalia,
but protrusion must be initiated by a centre, or centres, anterior to ganglion VIII, for
section of the nerve cord in front of this ganglion prevents it. However, once the genitalia have been protruded and copulation has begun, the genitalia seem to be maintained
in position solely by ganglion VIII, for section of the nerve cord immediately in
front of this ganglion during copulation does not cause the genitalia to be withdrawn.
Once copulation has begun spermatophore formation follows almost immediately,
and for the continuation of this, certainly up to the point at which the first reservoir
bladder becomes expanded, the union of the male and female genitalia must be
maintained. Formation is controlled again through ganglion VIII, which supplies
nerves to the accessory glands secreting the spermatophore, but the centre, or centres,
responsible for initiating the process and governing its continuation, certainly during
the earlier stages, must lie anterior to ganglion VIII, for formation is stopped by
section of the nerve cord between this ganglion and the anterior ganglia at any time
from the beginning of copulation until the expansion of the first reservoir bladder.
However, just as in copulation, the nervous connexion required between ganglion
VIII and the anterior parts of the nerve cord need be intact, certainly in the abdomen,
on one side of the ventral nerve cord only. Thus it would seem that the initiation of
spermatophore formation is brought about by the stimulus of copulation activating
an anterior centre which then initiates the secretion of spermatophore-forming
materials from the accessory glands by activating ganglion VIII. Just how the stimulus
of copulation is perceived is uncertain, but it may well be that the cerci play a major
part, though the possibility of chemoreception by the sensilla of the genitalia must
also be borne in mind.
Once the first reservoir bladder of the spermatophore has become expanded the
influence of the anterior centre is no longer required. The remaining stages of spermatophore formation seem to be controlled (in a purely reflex way) solely by ganglion
VIII, for they are stopped by the extirpation of this ganglion but are not affected by
inactivation of the cephalic ganglia, section of the ventral nerve cord or amputation
of the cerci. Not even the removal of the female, ending copulation, interferes with
the immediate continuation of spermatophore formation, but for the final stage of the
process, the extrusion of the spermatophore tube, the female must be present, though
her role seems then to be a purely passive one.
The sequence of male behaviour leading up to spermatophore formation thus
appears to be controlled and co-ordinated solely through the nervous system. The
possibility of the participation of hormonal systems cannot be ruled out, however,
for hormone material may be conveyed along the nerves, and it is possible that simple
Spermatophore formation in the African migratory locust
435
chemical stimuli, detected by the chemoreceptors of the mouthparts and genitalia,
may also be involved.
SUMMARY
1. The behaviour of the male culminating in spermatophore formation is divisible
into four phases: approach; mounting; copulation; and spermatophore formation.
All appear to be controlled and co-ordinated solely through the nervous system.
2. The initiation of sexual behaviour requires the brain and one or more of the
receptor-bearing head structures (antennae, palpi and eyes) to be functional.
3. Approach and mounting behaviour involves the receptors, nervous system and
effectors of the head and thorax only.
4. Copulation is controlled through the ganglia of the abdomen but for its initiation
these ganglia have to be in nervous communication with a centre anterior to them.
Also both cerci must be functional.
5. Spermatophore formation is initiated, and its early stages are controlled, by an
anterior centre which has its effect through the last abdominal ganglion. The later
stages of formation.appear to be controlled by the last abdominal ganglion alone.
6. The presence of the female is essential for the initiation and early stages of
spermatophore formation, but her role during the later stages of the process appears
to be a completely passive one.
My sincere thanks are due to Prof. James Brough, in whose Department the work
was done, to the Anti-Locust Research Centre, London, for supplying the original
locust material, and to Dr G. T. Jefferson and Dr P. T. Haskell for reading the
manuscript and for helpful suggestions.
REFERENCES
BOLDYREV, V. F. (1929). Spermatophore fertilization in the migratory locust (Locusta mtgratoria L.).
Izv. prikl. Ent. Leningrad, 4, 189-218.
GREGORY, G. E. (1965). The formation and fate of the spermatophore in the African migratory locust,
Locusta migratoria migratorioides Reiche and Fairmaire.
HASKELL, P. T. (1956). Hearing in certain Orthoptera. I. Physiology of sound receptors. J. Exp. Biol.
33, 756-66.
HASKELL, P. T. (1962). Sensory factors influencing phase change in locusts. Colloques Int. Cent. Nat.
Rech. Set. 114, 1-14.
HUBER, F. (1953). Verhaltensstudien am Mfinnchen der Feldgrille (Gryllus campettris L.) nach Eingriffen am ZentralnervensyBtem. Verh. dtich. zool. Get. 1952, 138—49.
HUBER, F. (1955). Sitz und Bedeutung nervoser Zentren ftir Instinkthandlungen beim Mannchen
von Gryllus campestris L. Z. Tierpsychol. ia, 12-48.
KARLBON, P. & BUTENANDT, A. (1959). Pheromones (ectohormones) in insects. Ann. Rev. Ent. 4, 39-58.
LOHER, W. (i960). The chemical acceleration of the maturation process and its hormonal control in
the male of the desert locutt. Proc. Roy. Soc. B, 153, 380-97.
MIKA, G. (1959). Uber das Paarungsverhalten der Wanderheuschrecke Locuita mtgratoria R. und F.
und deren AbhSngigkeit vom Zustand der inneren Geschlechtsorgane. Zool. Beitr., Berl., 4, 153203.
ROEDER, K. D. (1935). An experimental analysis of the sexual behaviour of the praying mantis {Mantis
rdigiosa L.). Biol. Bull., Woods Hole, 69, 203-20.
WALLACE, G. K. (1958). Visual perception and behaviour in the desert locust. Ardm. Behav. 6, 242-3.
WALLACE, G. K. (1959). Visual scanning in the desert locust Schistocerca gregaria Forskal. J. Exp. Biol.
36, 512-25.