f. lnsec! Yhysiol. Vol. 30, No. 4. pp. 271-278, 1984 0022-1910/84 $3.00+0.00

Printed in Great Britain. All rights reserved Copyright 0 1984 Pergamon Press Ltd

NEURAL INHIBITION OF EGG-LAYING IN THE LOCUST, LOCUSTA MIGRATORIA

ANGELA B. LANGE, IAN ORCHARD* and BARRY G. LOUGHTON Department of Biology, York University, 4700 Keele Street, Downsview, Ontario, M3J lP3 and *Department of Zoology, University of Toronto, 2.5 Harbord Street, Toronto, Ontario, M5S 1Al Canada

{Rece~zled 4 August 1983; revised 15 September 1983)

Abstract-The role of the oviducal nerves during egg-laying in Locusra ~~g~a~5~~u has been examined. Section of the oviducal nerves did not inhibit egg-laying in any observable way. Electrical stimulation of the oviducal nerves resulted in a contraction of the common and lower lateral oviducts which propelled ovulated eggs up towards the ovaries. Recordings from oviducal nerves using chronically implanted electrodes showed that electrical activity was low during actual egg-laying, but high at times when egg-laying was not occurring (i.e. during digging behaviour, or_following interruption of egg-laying). During these periods of high activity recurrent bursts of action potentials occurred. Similar patterns of electrical activity were recorded in semi-intact preparations using suction electrodes applied to exposed oviducal nerves of locusts which had been interrupted during the process of egg-laying. High frequency bursts of activity were recorded simultaneously from both left and right oviducal nerves.

It is concluded that one function of the oviducal nerves is to inhibit egg-laying at inappropriate times, by inducing contractions of the oviducts which propel eggs back towards the ovaries. These nerves therefore provide a physiological basis for part of the adaptive ovipositional activities of locusts.

Key Word In&x: Locusta. egg-laying, neural inhibition

INTRODUCTION

Egg-laying procedures vary widely among insects (review by Engelmann, 1970). Only a few species deposit their eggs at random, while most show adap- tive oviposition behaviour, retaining their ovulated eggs within the lateral oviducts until a suitable ovi- position site is found. As a result ovulation is fre- quently separated fr,m oviposition by a period of egg storage. This process occurs in locusts where mated females develop successive batches of eggs at approx- imately 5-day intervals. Mature eggs are “ovulated” from the ovaries and remain within the lateral ovi- ducts until they are laid. During this latter event the eggs must progress from the lateral oviducts into the common oviduct before being faid as a pod. Locusts are site selective and generally only deposit their egg pods into moist, loose soil (Kennedy, 1949) and will even abandon a site if it proves too dry or sodden. This assures protection of the eggs from desiccation, predators, parasites or decay. However, the phys- iological mechanism whereby eggs are retained within the lateral oviducts has not been described.

Egg-laying in many insects is stimulated by mating (see reviews by Leopold, 1976; Gillott and Friedel, 1977) and the physiological mechanisms associated with this stimulation appear to be almost exclusively centred around the neuroendocrine system. Thus, it has generally been proposed that mating influences the release of a hormone which enhances myogenic contractions of the oviduct muscles, resulting in egg progression atong the oviducts. Such hormonal con- trol of egg-laying has been proposed in locusts (High- nam, 1962, Okelo, 1971).

However, since insect oviducts can be innervated (Hartmann and Loher, 1974; Thomas, 1979; Cook et

al., 1980) the possibility of both nervous and hor- monal control of egg-laying exists. In spite of this. the neural control of egg-laying has only been studied in Carausius morosus (Thomas and Mesnier, 1973; Thomas, 1979) where, on the basis of nerve section experiments, it has been proposed that progression of eggs into the common oviduct is regulated by the nervous system.

Recently we have observed that even though the muscles of the oviducts of Locusta possess a myo- genie rhythm (Lange et al., 1983) which may be under hormonal control (Girardie and Lafon-Cazal, 1972) they are also innervated. Furthermore, we detected bursts of electrical activity within the oviducal nerves which resulted in contractions of the lower lateral and common oviducal muscles (Lange er al., 1983). This type of electrical activity was obtained from locusts which had been interrupted while in the process of egg-laying and not from other female locusts. Since our interests lay in the physiological mechanism of egg-laying in locusts, these observations led us to consider two possible functional explanations for the nervous innervation. The first was that the con- tractions induced by the electrical activity were in- volved with egg expulsion; the second that they were involved in the mechanism for egg retention, thus preventing egg release at inappropriate times. The present study provides evidence in favour of the latter of these two hypotheses.

MATERIALS AND METHODS

Locusta m~grat~r~a were reared under crowded conditions on a 12 h light: 12 h dark regime and fed on freshly grown wheat and bran. The temperature in the cages rose to 36°C during the photophase and fell

272 ANGELA B. LANGE et al.

to 23°C during the scotophase. Under these condi- tions mature mated females laid egg pods approxi- mately every 5 days.

Kert!e section

In order to sever the pair of oviducal nerves which pass from the seventh abdominal ganglion to the oviducts (Lange et al., 1983), a small flap of cuticle was cut in the seventh abdominal sternite and folded back to expose the oviducts (as in Fig. 1). The oviducal nerves leading to the oviducts were cut, the flap of cuticle replaced and the wound sealed with wax. Sham-operated controls were treated similarly but their nerves were left intact. Six experimentals and 6 controls were treated in this way.

In order to record on-going electrical activity from the oviducal nerves of semi-intact preparations, locusts were anaesthetized with CO,. The oviducts were exposed by a mid-ventral incision and the preparation was flooded with physiological Ringer (Hoyle, 19.55). En-passant recordings were made from either one or both of the oviducal nerves using glass suction electrodes. Signals were amplified ( x 1000) by means of a WP instrument DAM SA differential amplifier (band pass filters set at 300 Hz and 1 KHz), displayed on a Tektronix storage oscil- loscope and concurrently taped on a Vetter FM recorder for subsequent playback and analysis.

Electrical stimulation of the oviducal nerves was performed in citro. The ovaries and oviducts with their oviducal nerves were dissected from ovulated locusts (i.e. locusts which had mature eggs within their lateral oviducts). The preparation was placed in Ringer and attached to the wax base of a Perspex

FB

T

iov

Fig. 1. Diagram to illustrate the attachment of recording electrodes used in the free-walking insect preparation. A window is cut in the ventral surface of the abdomen through the seventh abdominal sternite (VII) to reveal the lateral oviducts (LOV) extending from the common oviduct (COV). The oviducal nerves (OVN) which innervate the lower lateral oviducts and anterior common oviduct have been exaggerated for clarity. The recording electrode (E) encircles the nerves as well as the fat body (FB) and trachea (T) between them. These additional structures add support. The flap is then replaced and sealed, along with the elec- trodes. with wax. The Roman numerals indicate the 6th and

7th abdominal sternites.

chamber with the aid of insect pins. The oviducal nerves were then electrically stimulated via a suction electrode using 0.5 ms square wave pulses delivered through a Grass Instruments SD9 stimulator. The resulting effects were photographed through a dis- secting microscope.

Electrode implantation

For implantation, females were removed from the sand shortly after digging behaviour had begun. We have observed that female locusts interrupted in this manner cease egg-laying but will resume shortly after being provided with a suitable oviposition site. Thus we were able to implant electrodes and have these same locusts lay eggs within several hours. A small fIap of cuticle was cut in the seventh abdominal sternite and folded back to expose the oviducts (see Fig. 1). Recordings were made from oviducal nerves of animals in the process of egg-laying, using im- planted electrodes similar to those used by Runion and Usherwood (1966) (Fig. 1). Forty-gauge insulated nickel or copper wire was cut into two 30 cm lengths which were then twisted about each other. An area close to the tips of the 2 wires of this electrode was stripped of insulation on one side only for a distance of about 1 mm. The stripped parts of this electrode were crimped around the oviducal nerves (which had been exposed as if for nerve section) of locusts which had been interrupted during digging. The electrode was then led out of the body cavity and held in position by replacing the flap of cuticle and sealing with wax. Initial trials revealed the nerves to be fairly fragile and better success was obtained when the strip of fat body lying between the 2 nerves was included within the electrode (Fig. 1). Since abdominal exten- sion for egg-laying occurs between segments 5 and 6 (Jorgensen and Rice, 1983), the operation of elec- trode implant through segment 7 did not interfere with this process. It also meant that the abdominal extension did not interfere with the recording elec- trode.

Implanted electrodes of this type allowed record- ings to be made from an intact free-moving insect. These insects were allowed to lay egg pods into moistened sand between two narrowly spaced glass plates. In this way it was possible to record electrical activity from the oviducal nerve of an intact locust and at the same time observe eggs being deposited.

RESULTS

Figure 2a is a record of electrical activity from the exposed oviducal nerves of mature female locusts. Small amplitude action potentials of low, continuous frequency are evident. In contrast, the patterning of electrical activity recorded from female locusts which had been interrupted during egg-laying consisted of both large and small amplitude action potentials occurring in repetitive bursts. The frequency of action potentials within a large amplitude burst reached 20-30 Hz while the small amplitude burst frequency was greater than 40Hz (Figs 2b and 3). The inter- burst interval which was constant within a prepara- tion, varied between locusts from 2 to 25s. Simulta- neous recordings from the 2 oviducal nerves revealed that similar activity occurred in both nerves concur-

Neural inhibition of egg-laying 273

preparations of this type. Figure 5a shows the elec- trical activity in the oviducaf nerves of a female locust in the process of digging into sand. Analysis of the waveform and duration of these potentials revealed them to be neural in original and not muscular. As can be seen, electrical activity was high during dig- ging (Fig. 5a) with one or two units showing bursting activity. On the other hand, when the abdomen was fully extended and the locust in the process of egg-laying, electrical activity within these oviducal nerves was minimal (Fig. 5b). In this case we ob- served the eggs through the glass walls of the egg- laying chamber as they were being laid. As can be seen in Fig. 5b, no electrical activity was associated with the deposition of an egg (arrow in Figure).

Another example is shown in Fig. 6 in which we observed effects of interruption of egg-laying upon

Fig. 2. Extracellular recordings. using a suction electrode. from the oviducal nerves in semi-intact preparations. A. A mature female locust which had not been in the process of egg-laying prior to dissection. Relatively continuous activity in the form of small amplitude action potentials is evident. B. An individual which had been interrupted during egg- laying. Note the recurring bursting pattern of both large and small amplitude action potentials. Similar results were ob- tained from at least 10 preparations. Scale bars: SO pV, Is.

rently (Fig. 3). Closer examination, however, showed that the action potentials from both nerves did not occur on a 1: 1 basis (Figs 3b, c) suggesting that each nerve is probably served by its own set of motor neurones which are closely, but not identically, coupled.

The first indication that the oviducal nerves were not absolutely necessa.ry for egg-laying came from the nerve section experiments. Section of the oviducal nerves did not inhibit egg laying in any way or result in any signi~cant difference in the number of eggs laid per pod. Thus, 36.5 _t 2.04 eggs were laid per pod by sham-operated while 34.7 k 1.4 eggs were laid per pod by “nerve sectioned” locusts.

With a view to further elucidating the role of these nerves, we examined the effects of electrical stimu- lation upon the ovidu.cts in Mv. The results of such an experiment are shown in Fig. 4. Electrical stimu- lation of the right oviducal nerve at 20 Hz resulted in a sustained contraction of the lower lateral and upper common oviduct which propelled the ovulated eggs of the right lateral oviduct back towards the ovaries (Fig. 4b). Upon cessation of stimulation, the eggs within the lateral oviduct returned to their pre- stimulatory position.

Recording from oviducal nerves with chronically implanted electrodes in mobile locusts supplied fur- ther information about the functional role of these nerves. Successful recordings were made from six

274 ANGELA B. LANGE el uI.

Fig. 5. Recordings from oviducal nerves in a free-walking locust using implanted electrodes show a high amount of electrical activity in a female locust in the process of digging into sand (A) and a minimal amount of electrical activity in the same female which was egg-laying (B). The electrical activity seen in the oviducal nerves during digging did not correspond to any movements seen in the ovipositor valves at this time. The arrow in B indicates the time at which an egg was seen being deposited. Note the absence of electrical activity associated with the event. Scale bars: A and B,

IOO~V, 0.5s.

electrical activity. Again the oviducal nerves showed high electrical activity while digging was in progress (Fig. 6a). This activity was reduced during egg-laying (Fig. 6b) but resumed when the locust was physically removed from the sand and ceased egg-laying (Figs 6c, d, e). High activity persisted for the 60min that the locust was deprived of an oviposition site. The pattern of activity, however, was not constant. Within 2min there were large amplitude action potentials in recurrent bursts, while after 15 min the activity was relatively constant, only to be replaced by bursting activity again by 60min (Figs 6c, d, e). When this locust was provided with sand she imme- diately dug in again and began to lay eggs, Activity was minimal at this time (Fig. 6f). Again the locust was manually removed from the sand and high electrical activity was seen (not shown). The same result was observed in several preparations. High electrical activity, in a recurrent bursting pattern, also occurred if the locust was disturbed and spontane- ously abandoned her oviposition site. Thus electrical activity in the oviducal nerves coincided with the cessation of egg-laying.

DISCUSSION

The results of the present paper clearly show that a function of the oviducal nerves in Locusta is to

prevent the passage of eggs out of the lateral oviducts during digging behaviour or when the locust is inter- rupted during egg-laying. Regular bursting patterns of electrical activity were recorded concurrently from both oviducal nerves of locusts interrupted during egg-laying. This pattern was recorded in semi-intact preparations, as well as from free-walking locusts using implanted electrodes. These locusts ceased egg- laying following interruption and so the intense elec- trical activity occurred at a time when eggs were not being laid. A similar pattern was evident in locusts during digging, again at a time when eggs were not being laid.

It has previously been shown that the bursting activity seen in the oviducal nerves results in sus- tained contractions of the lower lateral and common oviducal muscles (Lange et al., 1983). We now show that these contractions force mature eggs anteriorly away from the common oviduct (see Fig. 4). In the intact locust these contractions would hold the eggs in the anterior portion of both lateral oviducts, due to the simultaneous bursting activity of the left and right oviducal nerves.

The oviducal nerves are, in fact relatively silent during the actual process of egg-laying and clearly these nerves play little or no role for this particular event. The results of the nerve section experiment further contlrmed this, because locusts with severed oviducal nerves were still capable of laying eggs, Whether or not the nerves also play a role following ovulation and prior to ovipositional activities has not been examined in this paper. Thus we are not claim- ing that the only mechanism for retaining eggs within the lateral oviducts is through the oviducal nerves. For example, it has been found (Lange, unpublished observation) that the epithelial layer within the ante- rior portion of the common oviduct is highly con- voluted, thereby forming an obstruction to the pas- sage of eggs. These convolutions (and consequently the obstruction) would disappear upon extension of the common oviduct, as would occur with the abdominal stretching found during digging.

It is interesting to note that in the grasshopper Chorthippus curtipennis, Hartmann and Loher (1974) observed that artificial eggs injected into the lateral oviducts of females which had recently laid eggs, were pushed anteriorly up the lateral oviducts by muscular contraction. Presumably these muscular contractions could be under neural control in a similar manner to that observed in Locusta, and result in the retention of eggs until oviposition is required.

Nerve-section experiments have also implicated the nervous system in the control of egg-laying in the stick insect Car~sius morosus (Thomas, 1979: Thomas and Mesnier, 1973). In this insect, egg progression from the lateral into the common oviduct is prevented by contraction of the muscles of the common oviduct and it is believed that these muscles are controlled by nerves from the 7th abdominal ganglion (Thomas, 1979). This mechanism prevents eggs from being deposited at an inappropriate time, which in ~arausius appears to be during the photo- phase. When an egg is ovulated during the day it remains in a chamber located between the genital valves until the evening (Thomas, 1979). Sense organs in the wall of this chamber initiate a nervous reflex

Fig. 4. Electrical stimulation of an oviducal nerve results in contraction of the lower lateral and upper common oviduct. In A, the right oviducal nerve is within the suction electrode but in the absence of electrical stimulation the mature eggs can be seen in both lateral oviducts. In B, stimulation of the right oviducal nerve (0.5 ms pulses at 20 Hz) resulted in the mature eggs in the right lateral oviduct being forced anteriorly. L, left lateral oviduct; R, right lateral oviduct; E, suction electrode. Arrow denotes entry of

right oviducal nerve into suction electrode.

275

Neural inhibition of egg-laying

Fig. 6. Electrical activity of the oviducal nerves using implanted electrodes. Electrical activity was seen during digging (4) with little activity evident during egg-laying (B). High electrical activity returned after the locust was interrupted during egg-laying (C, D and E). A bursting pattern of electrical activity was seen 2 min after interruption (C). This activity became constant at I5 min (D) but returned to its bursting pattern by 60 min (E). When the locust was provided with a suitable oviposition site and the locust began

egg-laying once more, electrical activity was again minimal (F). Scale bars: 100 QV, 0.5 s.

via the 7th and 8th abdominal ganglia which results in a contraction of the common oviduct. Thus an egg can only pass into the common oviduct when the egg-chamber is empty. This would appear to be an adaptive measure to allow for the retention of eggs during the day. The diurnal rhythm of egg laying, is in addition, ensured by the release of a neuro- hormone (Thomas and Mesnier, 1973) which acts upon the genital valves. Thus in Curausius there is dual control of egg-laying in which egg progression is inhibited by the nervous system and egg expulsion by the genital valves depends upon a neuroendocrine factor (Thomas, 1969: Thomas and Mesnier, 1973). In a similar fashion, it has been proposed in locusts that during pre-ovipositional activities a hormone is released into the haemolymph. in anticipation of egg-laying, and this hormone increases the force of contractions of the muscles of the lateral oviducts (Okelo, 1971; Girardie and Lafon-Cazal, 1972). As a consequence, it is pertinent during the time when the hormone is present and the eggs are being propelled down the lateral oviducts, to possess a mechanism for inhibiting egg progression until the appropriate time. This would be necessary during pre-ovipositional behaviour (digging) an.d fohowing interruption while actually egg-laying.

Mating is believed to provide the stimulus for egg-laying in a number of insects (see Leopold, 1976; Gillott and Friedel, 1977) with the active factor being produced by the male accessory reproductive glands. While most studies have concentrated on examining the influence of mating on the release of neuro- hormones which may control movement of eggs within the genital tract. it would seem relevant for mating to activate motor programmes which control the behaviour associated with oviposition, However, while a number of reports have dealt with the local- ization of the control centre for oviposition within the central nervous system, few studies have examined the possible action of accessory gland secretions upon these centres (Leopold, 1976). Yamaoka and Hirao (1977) showed that injection of extracts of male reproductive tracts into virgin moths of Bombyr mori resulted in stimulation of egg-laying, and an increase in the spontaneous activity of motor neurones in the last abdominal ganglion. These motor neurones were also activated by direct application of this extract to isolated preparations of the last abdominal ganglion (Yamaoka, 1977). While these motor neurones inner- vate the genital organs and the abdominal muscular system, their function in oviposition was not deter- mined. Nevertheless it would appear that male secre-

278 ANGELA B. LANGE et al.

tions may induce the activation of a programme associated with oviposition.

Decapitation has often been observed to induce an egg-laying reflex (see Leopold, 1976). The appearance of oviposition posturing upon isolation of the abdo- men in locusts (Vincent, 197.54, suggested that the circuitry for the behaviour resides largely in the abdominal ganglia and that this behaviour is under anterior inhibitory control. The control of adaptive ovipositional ~hav~our (i.e. the orderly deposition of eggs within a suitable substrate) may overlap the mechanism concerned with movement of eggs within the genital tracts (Leopold, 1976). Indeed in S&is- tocerca, Leahy (1973) found that injection of acces- sory glands into virgin females increased the rate of oviposition, but also reduced the tendency for scat- tered eggs. Thus we may anticipate that the motor programme involved in preventing egg progression in Locusta may be primed by mating and activated at the appropriate time by internal stimuli, During times when egg-retention is required this motor programme must be activated and is manifested as a rhythmic pattern of bursting activity in both oviducal nerves. The action potentials do not occur on a 1 : 1 basis between nerves, however, and so each nerve probably possesses its own set of motor neurones. The overall pattern in each oviducal nerve is very similar indeed, implying that both sets of motor neurones are being driven by similar if not identical input or, once triggered, they are weakly coupled. At the present time we know little of the motor neurones or inter- neurones involved in generating this rhythm. How- ever, the preparation would appear to provide a useful system for examining such a programme, and for identifying the trigger mechanisms involved and the influence of mating. Work towards this end is currently in progress.

~cknou,ledge~ent-This work was supported by the Natu- ral Sciences and Engineering Research Council of Canada. We are most grateful to Professor Werner Loher for support and encouragement during this work. and for reading an early draft of the manuscript.

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