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Abdominal stretch reception in Dipetalogaster maximus (Hemiptera: Reduviidae)
J. Insecr Physiol. Vol. 30. No. 8. pp. 629-633. Printed
in Great
Britain.
All nghts
1984 Copyright
reserved
0022-1910~84 S3.00 + 0.00 ‘t’ 1984 Pergamon Press Ltd
ABDOMINAL STRETCH RECEPTION IN DIPETALOGASTER MAXIMUS (HEMTPTERA: REDUVIIDAE) H. F. Department
of Zoology,
Duke
NIJHOUT
University.
Durham.
North
Carolina
27706, U.S.A.
Abstract-Each half abdominal segment in Sth-instar larvae of the giant bloodsucking reduviid, Dipetalogasrer ma.uimus, contains 3 stretch receptor neurones. one associated with the tergosternal muscles, one with the ventral intersegmental muscles and one with the dorsal intersegmental muscles. Each of the three receptors respond phasically to the onset of stretch in its respective muscle group, but none show persistent activity upon prolonged stretch. By contrast. stretch of the main abdominal nerves (which run between the thoracic ganglion and the ventral intersegmental muscles of each abdominal segment) is accompanied by a prolonged and sustained pattern of discharge by an as yet unidentified neurone. the rate of discharge being proportional to the degree of stretch. In life. the abdominal nerves become stretched to about 145”. of their resting length when the larva takes a bloodmeal. Thus it appears that in Dipetalogusier stretch of the abdominal nerves themselves is the only mechanism for stretch reception after a blood meal. K~,J Word 1nde.x : Stretch hormone
receptors.
Rhodmus. Dipr/rthgrrtrw, prothoracicotroplc
hormone,
diuretic
release of a diuretic hormone (Maddrell. 1963. 1964b). In d series of elegant experiments Maddrell (1964b) showed that diuresis is only provoked by vertical distention of the abdomen. He concluded that the stretch receptors involved in stimulating diuresis must lie in the vertical tergosternal muscles (in the lateral region of the abdomen) and not in the intersegmental muscles. While a single bloodmeal provokes secretion of both PTTH and diuretic hormone. these two hormones are secreted over a very different time course. Stretch-stimulated secretion of PTTH occurs for a period of 3-5 days (the critical period of Wigglesworth [ 19341). Nerve section experiments have shown that continuous activity of the stretch receptors during this period is apparently required (Wigglesworth, 1934). By contrast, stretch-stimalated secretion of diuretic hormone occurs only for a period of 3 h. though continuous stimulation during this period is also necessary (Maddrell, 1964a,b). This radical difference in temporal response to stretch could be most readily explained if different stretch receptors are involved in each response. The receptor that controls PTTH release should not adapt for several days, while the receptor that controls diuretic hormone secretion should adapt in a few hours or should be so located that it is no longer effectively stretched after 4&50”;, of the meal volume has been eliminated by diuresis. A particularly intriguing feature is that the dorsal and ventral abdominal surfaces of adult Rhodnius are heavily sclerotized and the abdomen does not elongate with a bloodmeal (Maddrell. 1964b). Thus adults stretch only in the vertical dimension during feeding and only the tergosternal muscles can be stretched. Since adults diurese after a bloodmeal but do not secrete PTTH (Wigglesworth. 1970) it seems possible that tergosternal muscle recep-
INTRODUCTION
At regular intervals during an insect’s larval life. prothoracicotropic hormone (PTTH), produced by neurosecretory cells in the brain. is released into the circulatory system and stimulates the prothoracic glands to secrete the moulting hormone. edysone. The bloodsucking Hemiptera of the Family Reduviidae are among the very few insects in which we know the first step in the stimulation of PTTH secretion (see Nijhout [I9811 for a review). thanks to the classical work of Wigglesworth (1934, 1936) on Rhodnius prolixus. The brain of larvae of Rhodnius begins to secrete PTTH shortly after the animal takes a full bloodmeal. The release of PTTH can be inhibited by cutting the abdominal nerves or by severing the ventral nerve cord anterior to the thoracic ganglionic mass. On the basis of these findings Wigglesworth postulated that stretch receptors in the abdomen are activated when the animal engorges and then send impulses to the brain that activate the brain’s neurosecretory system to secrete PTTH. Anwyl (1972) has investigated the structure and properties of an abdominal stretch receptor in Rhodnius. He verified the earlier observations of Osborne and Finlayson (1962) that Rhodnius (like other Hemiptera studied so far) does not possess classical stretch receptors. Functional stretch reception, however, is provided by certain multiterminal peripheral neurones associated with the dorsal intersegmental muscles. Anwyl (1972) studied the physiology of these neurones in isolated preparations and showed that they responded to longitudinal stretch of the abdomen. At maximal stretch their adapted discharge frequency was twice the unstretched firing frequency. Stretch reception after a bloodmeal not only provokes the secretion of PTTH but also causes the 629
H. F. NIJHOUT
630
tors are involved in the control of diuretic hormone (although the actual existence of tergosternal stretch receptors has not yet been critically demonstrated) whereas the dorsal intersegmental receptors of Anwyl may control PTTH secretion. The present paper reports on a study of abdominal stretch reception in the giant blood-sucking reduviid, Dipetaloguster muximus. Except for its size, the gross anatomy and phsyiology of this species is virtually identical to that of Rho&k. Dipetalogaster is the largest reduviid known (Ryckman and Ryckman. 1967). adults measuring 4cm in length and each instar being about 6 times larger than the corresponding one in Rho&Gus. By virtue of its large size. Dipetaloguster is ideally suited for neurophysiological experiments-experiments that can be performed on Rhodnius only with considerable difficulty due to the constraints of size and space. MATERIALS
AND
METHODS
Colonies of Dipetaloguster rnusirnus were maintained at a constant temperature of 27 C under a long-day ( 16L: 8D) photoregime. All experiments described in this paper were carried out on 5th (final)-instar nymphs 3-6 weeks after moulting. Dissections and neurophysiological recordings were done on specimens submerged in Rhodnius saline (Maddrell and Phillips. 1975). Neuroanatomy was studied in wholemounts, using vitally stained methylene blue preparations. Methylene blue, however. stained very erratically. hence neuroanatomy was also studied in fixed wholemounts using Mann’s stain (methyl1917). Monopolar neublueleosin; Buxton,
rophysiological recordings were made using a tungsten hook electrode. All recordings were made from nerves of the 3rd abdominal segment. In each preparation, all branches of the nerves not entering the muscle group in question, or continuing beyond to other muscles or to cuticular sensilla. were cut. Isolated preparations of dorsal and ventral intersegmental muscles consisted of pieces of integument at least 3 segments long (segments 2, 3 and 4) with muscles and epidermis intact. One side of the preparation was held in a fixed clamp submerged in a shallow. saline-filled trough. This clamp also served as the earth electrode. The opposite side of the preparation was attached to a clamp that could be moved back and forth by a micromanipulator. Tergosternal muscles were stretched in half-abdomens by clamping the ventral midline in the stationary clamp and the dorsal midline in the clamp mounted on the micromanipulator. Stretch thus consisted of separating the dorsal and ventral surfaces of the abdomen at the body midline. Stretch was monitored and recorded by means of a Linear Variable Differential Transformer (LVDT, Pickering & Co.) coupled to the micromanipulator. RESULTS
Anatomy
of the ahdominul
newous
.sy.vtcrn
The gross anatomy of the abdominal musculature and its innervation in the 3rd abdominal segment of a Dipetaloguster nymph is shown in Fig. I. Each half abdominal segment contains only 3 major groups of muscles. A set of 7 dorsal intersegmental muscles (DIM), two tergosternal muscles (TSM). and a set of
I
Imm
1
I
Fig. 1. Diagrammatic representation of the abdominal musculature and innervation of a Sth-instar nymph of Dipetalogastrr. The right half of the 3rd abdominal segment is shown. Numbered circles indicate the locations of cell bodies of putative stretch receptor neurones associated with each muscle group. Roman numerals identify branches of the abdominal nerves referred to in the text. DIM: dorsal intersegmental muscles. VIM: ventral intersegmental muscles. TSM tergosternal muscles. ,vp: spiracle.
631
Stretch reception in Dipetaloguster 9 ventral intersegmental muscles (VIM). Because of lateral fusion the number of DIM and VIM appears to vary slightly from individual to individual. The muscular anatomy of Dipetaloguster thus closely resembles that of Rhodnius prolixus (Maddrell. 1964b), the major difference being that Rhodnius possesses 3 TSM rather than 2 in each half abdominal segment. All muscles in each half abdominal segment are innervated by a single nerve (bearing both sensory and motor neurones) emanating from the thoracic ganglion and branching into several major trunks as it approaches the segment, as gross anatomy essentially identical to that described by Maddrell (1964b) for Rhodnius. Since there is no conventional nomenclature for abdominal nerves in reduviids, I have indicated the various branches of the third abdominal nerve by Roman numerals (Fig. I) and shall refer to them by these numbers throughout this paper. Hemiptera do not possess morphologically identifiable stretch receptors. Instead, functional stretch receptors are provided by certain peripheral multiterminal neurones which are not associated with a specialized connective tissue strand but whose dendrites penetrate muscles and come to lie parallel to the muscle fibres (Osborne and Finlayson, 1962; Anwyl. 1972). The location and properties of such a stretch receptor neurone associated with the DIM of Rhodnius has been described by Anwyl (1972). The cell body of this neurone is large and is situated within the “dorsal motor nerve” close to the fork at which this nerve branches from the “tergal nerve”. In Dipetaloguster I found three such nerve cell bodies in each half abdominal segment, one associated with each of the major muscle groups. As a rule these cell bodies were located at or near a fork in the nerve as indicated in Fig. 1. although their precise position varied between individuals. The cell body associated with the DIM is in a homologous location to the stretch receptor cell body in Rhodnius described previously by Anwyl (1972). The other two cells have not been previously described but their size, their location within a nerve and their proximity to a muscle are all characteristics shared with the neurone associated with the DIM in Rhodnius and in Dipetalogasrer. Physiological experiments described below demonstrate a stretch reception function for each of the three muscle groups and implicate the cell
._
-_
0.5mml I set
Fig. 2. Response of the DIM receptor to longitudinal stretch of the integument. Three stretch episodes of progressively longer duration are recorded. Lower trace in each case represents output of the LVDT measuring the degree of stretch. Scale bar represents stretch over an entire preparation between clamps (see Methods) which consists of a length of 2 segments or about 3.1 mm. unstretched.
Fig. 3. Response of the TSM receptor to two brief and one more prolonged periods of stretch. Lower traces are output of LVDT which measures separation of the dorsal and ventral surfaces of the abdomen at the abdominal midline. Since the TSM insertions are well distal to the midline, TSM’s were stretched to approx I ? to I i of the extent indicated by the LVDT trace.
bodies described neurones. Physiological
above as those of stretch
receptor
studies on stretch remptioil
The DIM receptor. Figure 2 illustrates the response of the DIM receptor of Dipefulogasfer to longitudinal stretch. The unstretched length of this preparation was 3.1 mm. A full bloodmeal would have stretched the cuticle of this preparation about I .2 mm. Recordings were made from the midpoint of nerve branch V (see Fig. I). The top panel shows the response, to two successive brief stretches and the bottom panel shows the response, in a different preparation. to a more prolonged episode of stretch lasting 6 s. In all cases the muscle receptor responds to the initiation of stretch with a brief burst of action potentials lasting 0.6-0.9 s. No tonic activity was observed during or subsequent to stretching, even in preparations in which stretch was maintained for several hours. nor was there any response to the cessation of stretch or to the reversal of stretch (relaxation). Evidently the DIM receptors of Dipetuloguster respond only to the onset of stretch. The TSM receptor. The response of the TSM receptor to dorsoventral stretch of the abdomen is shown in Fig. 3. Recordings were made from nerve branch IV (Fig. 1). Because of the mode of stretching (see Methods) the LVDT record measures separation of the dorsal and ventral surfaces of the abdomen at the body midline. Stretch at the level of the TSM’s is l to i of that indicated by the LVDT. A full bloodmeal normally stretches the TSM about 2.5-3 mm, depending on the size of the animal. The responses of this receptor to stretch are in all respects identical to those of the DIM receptor. The response is phasic. occurring only at the onset of stretch and the receptor shows neither background activity nor a persistent response to continuously-maintained stretch of the TSMs. The VIM receptor. The response of the VIM receptor differs significantly from that of the preceding two in that. while it responds phasically to longitudinal stretch of the VIM. it responds tonically and persistently to stretch of the main abdominal nerve (Fig. 4). Recordings were made from the midpoint of nerve branch II (Fig. 1). The record shown in Fig. 4a is that of an unstretched nerve exhibiting a low and somewhat erratic pattern of
632
H. F.
Fig.
4.
NIJHOUT
Response of the VIM receptor
onset of longitudinal
stretch
recorded from branch II (see Fig. 1) of the abdominal nerve. m. of integument with attached DIM only: II. onset of stretch of nerve branch II only.
discharges. Superimposed on this are two episodes of longitudinal stretch of the integument and the VIMs. The onset of stretch is marked by arrows (m). An LVDT was not used to monitor stretch in these preparations but the stretch regimens were similar to the first stretch episode shown in Fig. 3. It is clear from Fig. 4a that the response to muscle stretch is identical in character to that observed for the DIM and TSM receptors. The background activity pattern in this nerve is not affected by the episodes of muscle stretch. Figure 4b is a record from the same preparation as shown in Fig. 4a after nerve branch II (the nerve branch being recorded from) had been stretched to approx I l5”,, of its original length. When the nerve is maintained at this length and the integument plus the attached VIMs is stretched (arrows. m) one observes the usual pattern of phasic activity superimposed on the steady background level of discharge due to the stretched nerve. Stretch of the integument does not appear to affect or interact with the activity due to nerve stretching. It is also evident (Figs 4a,b) that the two responses observed are due to the activities of two different cells each with a characteristic amplitude of response. One of these cells responds only to stretch of the integument (presumably stretch of the VIMs), while the other responds only to stretch of nerve branch I I. Figure 4c shows a more prolonged record of the response of the nerve receptor to successive stretchings of the nerve (arrowsn), each lengthening the nerve by an additional 15:;. The enhanced rate of discharge routinely persisted undiminished for 8-10 h, the normal lifespan of these stagnant preparations. Measurements on the 3 preparations that were active for slightly longer than 10 h gave the following quantitative data. Prior to nerve stretch the discharge frequency was zero. Five minutes after a moderate stretch the mean discharge frequency was 28 impulses/s. This frequency remained undiminished for 5 h. Between 6 and 9 h there was a gradual decline in activity to a mean of 16 impulses/s. This decline in activity was accompanied by a significant decrease in the amplitude of the signal to about 40:; of its initial value. Whether the decline in activity observed after 5 h was due to adaptation is not known. The declining amplitude of the response and death of the preparation after 10 h suggest that at least some of the decline in activity was due to a deterioration of the preparation. No attempt was made to determine the numerical relationship between the degree of nerve stretch and the adapted
frequency of the response because it is unclear at present whether the entire nerve or only its terminal portion is responsive to stretch. Neither nerve branch III nor any other branch of the abdominal nerve showed a similar response to stretch, In order to determine whether or not this response to nerve stretch was physiological I measured the length of the abdominal nerve, between the point at which it exists the thoracic ganglion and the point at which branches II and III enter the VIM of the 3rd abdominal segment. in unfed and recently bloodfed larvae. Due to the enormous expansion of the abdomen after engorgement these segments of the abdominal nerve routinely become stretched to 145’:” of their length in an unfed larva. It is clear therefore that. in life, abdominal nerves become stretched to a significantly greater degree than was required to obtain the response illustrated in Fig. 4c. A subsequent search for additional nerve cell bodies within the abdominal nerves failed to disclose any, so that it is at present unclear whether the nerve cell body associated with the VIM is that of the cell responsible for the response to muscle stretch or to nerve stretch. Attempts to record the integrated response to abdominal stretch in intact animals, from the abdominal nerves immediately posterior to the thoracic ganglionic mass, have met with negligible success so far, because the records are swamped by high amplitude muscle potentials from the gut, which undergoes continuous peristalsis during and after a bloodmeal. DISCUSSION
It is evident from the responses of the VIM. DIM, and TSM receptors to stretch of the body wall that, with the proviso regarding the VIM receptor mentioned above, none of the three are likely candidates for stretch monitoring after a bloodmeal. All three receptors show a phasic response only, with no response at all to maintained stretch. These findings are in contrast to the observations of Anwyl (1972) on Rhodnius prolixus. Anwyl (1972) showed that the DIM of Rhodnius responds tonically to longitudinal stretch of the integument and, when stretched, shows a positive linear relationship between degree of stretch and discharge frequency of the receptor, with a maximal frequency of 16-18 impulses/s in a fully stretched preparation. This difference in stretch receptor behaviour between Rhodnius and Dipetulogastw appears to be real. In a series of pilot
633
Stretch reception in Dipeiulognr/er experiments on Rhodnius I have obtained substantially similar results to those that Anwyl has reported. That is, Rhodnius’ DIM receptors do indeed respond to stretch by a prolonged period of activity. However, in no case did this enhanced activity persist for more than 2&30 min. Anwyl (1972) did not report on the persistence of the enhanced activity of his preparations but one would expect that, in Rhodnius. receptors controlling diuretic hormone and PTTH ought to be active for at least 2 h and 4 days, respectively, as the experiments of Wigglesworth ( 1934) and Maddrell (1964a.b) strongly suggest. It therefore remains unclear whether the DIM receptor of Rhodnius is a viable candidate for the control of either hormone. Anwyl (1972) did not investigate the possible existence of other stretch receptors in Rhodnius. though Van der Kloot (1960. 1961) reported circumstantial evidence for the existence of stretch receptors in the TSM of this species. My own studies (unpublished) have revealed cell bodies embedded in the nerves that innervate the TSM and VIM of Rhodnius and confirm the presence of the cell body associated with the DIM (Anwyl, 1972). All three occur in homologous locations to those of Dipetalogusfrr (Fig. 1). In Dipetalogaster the only reasonable candidate for monitoring abdominal stretch after a bloodmeal appears to be nerve-stretch reception by nerve branch II. This nerve branch normally becomes stretched to 145”f0 of its normal length by abdominal swelling after a bloodmeal. Electrophysiological studies show that its discharge rate is proportional to the length to which the nerve is stretched (Fig. 4). Furthermore, a high rate of discharge of the stretched nerve continues for at least 10 h in isolated preparations. In Dipetatogastrr, diuresis after a bloodmeal persists for 6-7 h (Nijhout, unpublished), so that the activity of nerve II is sufficiently persistent to control the secretion of diuretic hormone. Whether or not it persists for the duration of PTTH secretion (the head-critical period is approx 6 days. Nijhout unpublished) is difficult to say. Since there appears to be only one tonic stretch receptor in each half abdominal segment, the possibility must exist that the time course of secretion of diuretic hormone and PTTH is controlled centrally rather than by the differential behav-
ior of two different populations as was suspected previously.
of stretch
receptors
Ackno~ledgc,ments~This work was supported, in part, by grants PCM 7680518 and PCM 7911779 from the National Science Foundation.
REFERENCES
Anwyl R. (1972) The structure and properties of an abdominal stretch receptor in Rhodnius proli.uus. J. hst~~ P/I\,.\ iol. 18, 2143-2154. Buxton B. A. (1917) On the protocerebrum of Micropter~.\(Lepidoptera). Trans. R. em. Sot. Lortd. 65, I lSl53. Maddrell S. H. P. (1963) Excretion in the blood-sucking bug. Rhodnius proli.ws Stal. 1. The control of diuresis. J. e.xp. Biol. 40, 247?256. Maddrell S. H. P. (1964a) Excretion in the blood-sucking bug, Rhodnius prokus Stal. II. The normal course of diuresis and the effect of temperature. J. c’.\-p. Bio(. 41, 163-176. Maddrell S. H. P. (1964b) Excretton m the blood-sucking bag. Rhodnius prolixus Stal. III. The control of the release of the diuretic hormone. J. exp. Biol. 41. 459472. Maddrell. S. H. P. and Phillias. J. E. (1975) Secretion of hypo-osmotic fluid by the iower Malpighian tubules of Rhodnius prollsus. J. esp. Biol. 62. 671-683. Nijhout H. F. (1981) Physiological control of molting in insects. .4m. Zool. 21, 631-640. Osborne M. P. and Finlayson L. H. (1962) The structure and topography of stretch receptors in representative orders of &cts. Q. JI Microsc. Sci. 103, 277-242. Rvckman R. E. and Rvckman A. E. ( 1967) Eoizootiologv of Trypanosoma &I in southwestern North America. Part X: The biosystematics of Dipetulogusrcr musimus in Mexico. J. med. En/. 4, 180-188. Van Der Kloot W. G. (1960) Neurosecretion in Insects. .% Rev. &I. 5, 35-52. Van Der Kloot W. G. (1961) Insect metamorphosis and Its endocrine control. Am. Zool. I. 3--9. Wigglesworth V. B. (1934) The physiology of ecdysis in Rhodnius proli.ws (Hemiptera). II. Factors controlling moulting and ‘metamorphosis’. Q, .I/. Mirmwi. Sci. 77. 19 l-222. Wigglesworth V. B. (lY36) The function of the corpus allatum in the growth and reproduction of Rhorlnius proli.ms (Hemiptera). Q. JI. Microsr. SC,;. 79, Yl- I2 I. Wigglesworth V. B. ( 1970) fnsecr Hornlonrs. I59 pp. Freeman. San Francisco.
in Great
Britain.
All nghts
1984 Copyright
reserved
0022-1910~84 S3.00 + 0.00 ‘t’ 1984 Pergamon Press Ltd
ABDOMINAL STRETCH RECEPTION IN DIPETALOGASTER MAXIMUS (HEMTPTERA: REDUVIIDAE) H. F. Department
of Zoology,
Duke
NIJHOUT
University.
Durham.
North
Carolina
27706, U.S.A.
Abstract-Each half abdominal segment in Sth-instar larvae of the giant bloodsucking reduviid, Dipetalogasrer ma.uimus, contains 3 stretch receptor neurones. one associated with the tergosternal muscles, one with the ventral intersegmental muscles and one with the dorsal intersegmental muscles. Each of the three receptors respond phasically to the onset of stretch in its respective muscle group, but none show persistent activity upon prolonged stretch. By contrast. stretch of the main abdominal nerves (which run between the thoracic ganglion and the ventral intersegmental muscles of each abdominal segment) is accompanied by a prolonged and sustained pattern of discharge by an as yet unidentified neurone. the rate of discharge being proportional to the degree of stretch. In life. the abdominal nerves become stretched to about 145”. of their resting length when the larva takes a bloodmeal. Thus it appears that in Dipetalogusier stretch of the abdominal nerves themselves is the only mechanism for stretch reception after a blood meal. K~,J Word 1nde.x : Stretch hormone
receptors.
Rhodmus. Dipr/rthgrrtrw, prothoracicotroplc
hormone,
diuretic
release of a diuretic hormone (Maddrell. 1963. 1964b). In d series of elegant experiments Maddrell (1964b) showed that diuresis is only provoked by vertical distention of the abdomen. He concluded that the stretch receptors involved in stimulating diuresis must lie in the vertical tergosternal muscles (in the lateral region of the abdomen) and not in the intersegmental muscles. While a single bloodmeal provokes secretion of both PTTH and diuretic hormone. these two hormones are secreted over a very different time course. Stretch-stimulated secretion of PTTH occurs for a period of 3-5 days (the critical period of Wigglesworth [ 19341). Nerve section experiments have shown that continuous activity of the stretch receptors during this period is apparently required (Wigglesworth, 1934). By contrast, stretch-stimalated secretion of diuretic hormone occurs only for a period of 3 h. though continuous stimulation during this period is also necessary (Maddrell, 1964a,b). This radical difference in temporal response to stretch could be most readily explained if different stretch receptors are involved in each response. The receptor that controls PTTH release should not adapt for several days, while the receptor that controls diuretic hormone secretion should adapt in a few hours or should be so located that it is no longer effectively stretched after 4&50”;, of the meal volume has been eliminated by diuresis. A particularly intriguing feature is that the dorsal and ventral abdominal surfaces of adult Rhodnius are heavily sclerotized and the abdomen does not elongate with a bloodmeal (Maddrell. 1964b). Thus adults stretch only in the vertical dimension during feeding and only the tergosternal muscles can be stretched. Since adults diurese after a bloodmeal but do not secrete PTTH (Wigglesworth. 1970) it seems possible that tergosternal muscle recep-
INTRODUCTION
At regular intervals during an insect’s larval life. prothoracicotropic hormone (PTTH), produced by neurosecretory cells in the brain. is released into the circulatory system and stimulates the prothoracic glands to secrete the moulting hormone. edysone. The bloodsucking Hemiptera of the Family Reduviidae are among the very few insects in which we know the first step in the stimulation of PTTH secretion (see Nijhout [I9811 for a review). thanks to the classical work of Wigglesworth (1934, 1936) on Rhodnius prolixus. The brain of larvae of Rhodnius begins to secrete PTTH shortly after the animal takes a full bloodmeal. The release of PTTH can be inhibited by cutting the abdominal nerves or by severing the ventral nerve cord anterior to the thoracic ganglionic mass. On the basis of these findings Wigglesworth postulated that stretch receptors in the abdomen are activated when the animal engorges and then send impulses to the brain that activate the brain’s neurosecretory system to secrete PTTH. Anwyl (1972) has investigated the structure and properties of an abdominal stretch receptor in Rhodnius. He verified the earlier observations of Osborne and Finlayson (1962) that Rhodnius (like other Hemiptera studied so far) does not possess classical stretch receptors. Functional stretch reception, however, is provided by certain multiterminal peripheral neurones associated with the dorsal intersegmental muscles. Anwyl (1972) studied the physiology of these neurones in isolated preparations and showed that they responded to longitudinal stretch of the abdomen. At maximal stretch their adapted discharge frequency was twice the unstretched firing frequency. Stretch reception after a bloodmeal not only provokes the secretion of PTTH but also causes the 629
H. F. NIJHOUT
630
tors are involved in the control of diuretic hormone (although the actual existence of tergosternal stretch receptors has not yet been critically demonstrated) whereas the dorsal intersegmental receptors of Anwyl may control PTTH secretion. The present paper reports on a study of abdominal stretch reception in the giant blood-sucking reduviid, Dipetaloguster muximus. Except for its size, the gross anatomy and phsyiology of this species is virtually identical to that of Rho&k. Dipetalogaster is the largest reduviid known (Ryckman and Ryckman. 1967). adults measuring 4cm in length and each instar being about 6 times larger than the corresponding one in Rho&Gus. By virtue of its large size. Dipetaloguster is ideally suited for neurophysiological experiments-experiments that can be performed on Rhodnius only with considerable difficulty due to the constraints of size and space. MATERIALS
AND
METHODS
Colonies of Dipetaloguster rnusirnus were maintained at a constant temperature of 27 C under a long-day ( 16L: 8D) photoregime. All experiments described in this paper were carried out on 5th (final)-instar nymphs 3-6 weeks after moulting. Dissections and neurophysiological recordings were done on specimens submerged in Rhodnius saline (Maddrell and Phillips. 1975). Neuroanatomy was studied in wholemounts, using vitally stained methylene blue preparations. Methylene blue, however. stained very erratically. hence neuroanatomy was also studied in fixed wholemounts using Mann’s stain (methyl1917). Monopolar neublueleosin; Buxton,
rophysiological recordings were made using a tungsten hook electrode. All recordings were made from nerves of the 3rd abdominal segment. In each preparation, all branches of the nerves not entering the muscle group in question, or continuing beyond to other muscles or to cuticular sensilla. were cut. Isolated preparations of dorsal and ventral intersegmental muscles consisted of pieces of integument at least 3 segments long (segments 2, 3 and 4) with muscles and epidermis intact. One side of the preparation was held in a fixed clamp submerged in a shallow. saline-filled trough. This clamp also served as the earth electrode. The opposite side of the preparation was attached to a clamp that could be moved back and forth by a micromanipulator. Tergosternal muscles were stretched in half-abdomens by clamping the ventral midline in the stationary clamp and the dorsal midline in the clamp mounted on the micromanipulator. Stretch thus consisted of separating the dorsal and ventral surfaces of the abdomen at the body midline. Stretch was monitored and recorded by means of a Linear Variable Differential Transformer (LVDT, Pickering & Co.) coupled to the micromanipulator. RESULTS
Anatomy
of the ahdominul
newous
.sy.vtcrn
The gross anatomy of the abdominal musculature and its innervation in the 3rd abdominal segment of a Dipetaloguster nymph is shown in Fig. I. Each half abdominal segment contains only 3 major groups of muscles. A set of 7 dorsal intersegmental muscles (DIM), two tergosternal muscles (TSM). and a set of
I
Imm
1
I
Fig. 1. Diagrammatic representation of the abdominal musculature and innervation of a Sth-instar nymph of Dipetalogastrr. The right half of the 3rd abdominal segment is shown. Numbered circles indicate the locations of cell bodies of putative stretch receptor neurones associated with each muscle group. Roman numerals identify branches of the abdominal nerves referred to in the text. DIM: dorsal intersegmental muscles. VIM: ventral intersegmental muscles. TSM tergosternal muscles. ,vp: spiracle.
631
Stretch reception in Dipetaloguster 9 ventral intersegmental muscles (VIM). Because of lateral fusion the number of DIM and VIM appears to vary slightly from individual to individual. The muscular anatomy of Dipetaloguster thus closely resembles that of Rhodnius prolixus (Maddrell. 1964b), the major difference being that Rhodnius possesses 3 TSM rather than 2 in each half abdominal segment. All muscles in each half abdominal segment are innervated by a single nerve (bearing both sensory and motor neurones) emanating from the thoracic ganglion and branching into several major trunks as it approaches the segment, as gross anatomy essentially identical to that described by Maddrell (1964b) for Rhodnius. Since there is no conventional nomenclature for abdominal nerves in reduviids, I have indicated the various branches of the third abdominal nerve by Roman numerals (Fig. I) and shall refer to them by these numbers throughout this paper. Hemiptera do not possess morphologically identifiable stretch receptors. Instead, functional stretch receptors are provided by certain peripheral multiterminal neurones which are not associated with a specialized connective tissue strand but whose dendrites penetrate muscles and come to lie parallel to the muscle fibres (Osborne and Finlayson, 1962; Anwyl. 1972). The location and properties of such a stretch receptor neurone associated with the DIM of Rhodnius has been described by Anwyl (1972). The cell body of this neurone is large and is situated within the “dorsal motor nerve” close to the fork at which this nerve branches from the “tergal nerve”. In Dipetaloguster I found three such nerve cell bodies in each half abdominal segment, one associated with each of the major muscle groups. As a rule these cell bodies were located at or near a fork in the nerve as indicated in Fig. 1. although their precise position varied between individuals. The cell body associated with the DIM is in a homologous location to the stretch receptor cell body in Rhodnius described previously by Anwyl (1972). The other two cells have not been previously described but their size, their location within a nerve and their proximity to a muscle are all characteristics shared with the neurone associated with the DIM in Rhodnius and in Dipetalogasrer. Physiological experiments described below demonstrate a stretch reception function for each of the three muscle groups and implicate the cell
._
-_
0.5mml I set
Fig. 2. Response of the DIM receptor to longitudinal stretch of the integument. Three stretch episodes of progressively longer duration are recorded. Lower trace in each case represents output of the LVDT measuring the degree of stretch. Scale bar represents stretch over an entire preparation between clamps (see Methods) which consists of a length of 2 segments or about 3.1 mm. unstretched.
Fig. 3. Response of the TSM receptor to two brief and one more prolonged periods of stretch. Lower traces are output of LVDT which measures separation of the dorsal and ventral surfaces of the abdomen at the abdominal midline. Since the TSM insertions are well distal to the midline, TSM’s were stretched to approx I ? to I i of the extent indicated by the LVDT trace.
bodies described neurones. Physiological
above as those of stretch
receptor
studies on stretch remptioil
The DIM receptor. Figure 2 illustrates the response of the DIM receptor of Dipefulogasfer to longitudinal stretch. The unstretched length of this preparation was 3.1 mm. A full bloodmeal would have stretched the cuticle of this preparation about I .2 mm. Recordings were made from the midpoint of nerve branch V (see Fig. I). The top panel shows the response, to two successive brief stretches and the bottom panel shows the response, in a different preparation. to a more prolonged episode of stretch lasting 6 s. In all cases the muscle receptor responds to the initiation of stretch with a brief burst of action potentials lasting 0.6-0.9 s. No tonic activity was observed during or subsequent to stretching, even in preparations in which stretch was maintained for several hours. nor was there any response to the cessation of stretch or to the reversal of stretch (relaxation). Evidently the DIM receptors of Dipetuloguster respond only to the onset of stretch. The TSM receptor. The response of the TSM receptor to dorsoventral stretch of the abdomen is shown in Fig. 3. Recordings were made from nerve branch IV (Fig. 1). Because of the mode of stretching (see Methods) the LVDT record measures separation of the dorsal and ventral surfaces of the abdomen at the body midline. Stretch at the level of the TSM’s is l to i of that indicated by the LVDT. A full bloodmeal normally stretches the TSM about 2.5-3 mm, depending on the size of the animal. The responses of this receptor to stretch are in all respects identical to those of the DIM receptor. The response is phasic. occurring only at the onset of stretch and the receptor shows neither background activity nor a persistent response to continuously-maintained stretch of the TSMs. The VIM receptor. The response of the VIM receptor differs significantly from that of the preceding two in that. while it responds phasically to longitudinal stretch of the VIM. it responds tonically and persistently to stretch of the main abdominal nerve (Fig. 4). Recordings were made from the midpoint of nerve branch II (Fig. 1). The record shown in Fig. 4a is that of an unstretched nerve exhibiting a low and somewhat erratic pattern of
632
H. F.
Fig.
4.
NIJHOUT
Response of the VIM receptor
onset of longitudinal
stretch
recorded from branch II (see Fig. 1) of the abdominal nerve. m. of integument with attached DIM only: II. onset of stretch of nerve branch II only.
discharges. Superimposed on this are two episodes of longitudinal stretch of the integument and the VIMs. The onset of stretch is marked by arrows (m). An LVDT was not used to monitor stretch in these preparations but the stretch regimens were similar to the first stretch episode shown in Fig. 3. It is clear from Fig. 4a that the response to muscle stretch is identical in character to that observed for the DIM and TSM receptors. The background activity pattern in this nerve is not affected by the episodes of muscle stretch. Figure 4b is a record from the same preparation as shown in Fig. 4a after nerve branch II (the nerve branch being recorded from) had been stretched to approx I l5”,, of its original length. When the nerve is maintained at this length and the integument plus the attached VIMs is stretched (arrows. m) one observes the usual pattern of phasic activity superimposed on the steady background level of discharge due to the stretched nerve. Stretch of the integument does not appear to affect or interact with the activity due to nerve stretching. It is also evident (Figs 4a,b) that the two responses observed are due to the activities of two different cells each with a characteristic amplitude of response. One of these cells responds only to stretch of the integument (presumably stretch of the VIMs), while the other responds only to stretch of nerve branch I I. Figure 4c shows a more prolonged record of the response of the nerve receptor to successive stretchings of the nerve (arrowsn), each lengthening the nerve by an additional 15:;. The enhanced rate of discharge routinely persisted undiminished for 8-10 h, the normal lifespan of these stagnant preparations. Measurements on the 3 preparations that were active for slightly longer than 10 h gave the following quantitative data. Prior to nerve stretch the discharge frequency was zero. Five minutes after a moderate stretch the mean discharge frequency was 28 impulses/s. This frequency remained undiminished for 5 h. Between 6 and 9 h there was a gradual decline in activity to a mean of 16 impulses/s. This decline in activity was accompanied by a significant decrease in the amplitude of the signal to about 40:; of its initial value. Whether the decline in activity observed after 5 h was due to adaptation is not known. The declining amplitude of the response and death of the preparation after 10 h suggest that at least some of the decline in activity was due to a deterioration of the preparation. No attempt was made to determine the numerical relationship between the degree of nerve stretch and the adapted
frequency of the response because it is unclear at present whether the entire nerve or only its terminal portion is responsive to stretch. Neither nerve branch III nor any other branch of the abdominal nerve showed a similar response to stretch, In order to determine whether or not this response to nerve stretch was physiological I measured the length of the abdominal nerve, between the point at which it exists the thoracic ganglion and the point at which branches II and III enter the VIM of the 3rd abdominal segment. in unfed and recently bloodfed larvae. Due to the enormous expansion of the abdomen after engorgement these segments of the abdominal nerve routinely become stretched to 145’:” of their length in an unfed larva. It is clear therefore that. in life, abdominal nerves become stretched to a significantly greater degree than was required to obtain the response illustrated in Fig. 4c. A subsequent search for additional nerve cell bodies within the abdominal nerves failed to disclose any, so that it is at present unclear whether the nerve cell body associated with the VIM is that of the cell responsible for the response to muscle stretch or to nerve stretch. Attempts to record the integrated response to abdominal stretch in intact animals, from the abdominal nerves immediately posterior to the thoracic ganglionic mass, have met with negligible success so far, because the records are swamped by high amplitude muscle potentials from the gut, which undergoes continuous peristalsis during and after a bloodmeal. DISCUSSION
It is evident from the responses of the VIM. DIM, and TSM receptors to stretch of the body wall that, with the proviso regarding the VIM receptor mentioned above, none of the three are likely candidates for stretch monitoring after a bloodmeal. All three receptors show a phasic response only, with no response at all to maintained stretch. These findings are in contrast to the observations of Anwyl (1972) on Rhodnius prolixus. Anwyl (1972) showed that the DIM of Rhodnius responds tonically to longitudinal stretch of the integument and, when stretched, shows a positive linear relationship between degree of stretch and discharge frequency of the receptor, with a maximal frequency of 16-18 impulses/s in a fully stretched preparation. This difference in stretch receptor behaviour between Rhodnius and Dipetulogastw appears to be real. In a series of pilot
633
Stretch reception in Dipeiulognr/er experiments on Rhodnius I have obtained substantially similar results to those that Anwyl has reported. That is, Rhodnius’ DIM receptors do indeed respond to stretch by a prolonged period of activity. However, in no case did this enhanced activity persist for more than 2&30 min. Anwyl (1972) did not report on the persistence of the enhanced activity of his preparations but one would expect that, in Rhodnius. receptors controlling diuretic hormone and PTTH ought to be active for at least 2 h and 4 days, respectively, as the experiments of Wigglesworth ( 1934) and Maddrell (1964a.b) strongly suggest. It therefore remains unclear whether the DIM receptor of Rhodnius is a viable candidate for the control of either hormone. Anwyl (1972) did not investigate the possible existence of other stretch receptors in Rhodnius. though Van der Kloot (1960. 1961) reported circumstantial evidence for the existence of stretch receptors in the TSM of this species. My own studies (unpublished) have revealed cell bodies embedded in the nerves that innervate the TSM and VIM of Rhodnius and confirm the presence of the cell body associated with the DIM (Anwyl, 1972). All three occur in homologous locations to those of Dipetalogusfrr (Fig. 1). In Dipetalogaster the only reasonable candidate for monitoring abdominal stretch after a bloodmeal appears to be nerve-stretch reception by nerve branch II. This nerve branch normally becomes stretched to 145”f0 of its normal length by abdominal swelling after a bloodmeal. Electrophysiological studies show that its discharge rate is proportional to the length to which the nerve is stretched (Fig. 4). Furthermore, a high rate of discharge of the stretched nerve continues for at least 10 h in isolated preparations. In Dipetatogastrr, diuresis after a bloodmeal persists for 6-7 h (Nijhout, unpublished), so that the activity of nerve II is sufficiently persistent to control the secretion of diuretic hormone. Whether or not it persists for the duration of PTTH secretion (the head-critical period is approx 6 days. Nijhout unpublished) is difficult to say. Since there appears to be only one tonic stretch receptor in each half abdominal segment, the possibility must exist that the time course of secretion of diuretic hormone and PTTH is controlled centrally rather than by the differential behav-
ior of two different populations as was suspected previously.
of stretch
receptors
Ackno~ledgc,ments~This work was supported, in part, by grants PCM 7680518 and PCM 7911779 from the National Science Foundation.
REFERENCES
Anwyl R. (1972) The structure and properties of an abdominal stretch receptor in Rhodnius proli.uus. J. hst~~ P/I\,.\ iol. 18, 2143-2154. Buxton B. A. (1917) On the protocerebrum of Micropter~.\(Lepidoptera). Trans. R. em. Sot. Lortd. 65, I lSl53. Maddrell S. H. P. (1963) Excretion in the blood-sucking bug. Rhodnius proli.ws Stal. 1. The control of diuresis. J. e.xp. Biol. 40, 247?256. Maddrell S. H. P. (1964a) Excretion in the blood-sucking bug, Rhodnius prokus Stal. II. The normal course of diuresis and the effect of temperature. J. c’.\-p. Bio(. 41, 163-176. Maddrell S. H. P. (1964b) Excretton m the blood-sucking bag. Rhodnius prolixus Stal. III. The control of the release of the diuretic hormone. J. exp. Biol. 41. 459472. Maddrell. S. H. P. and Phillias. J. E. (1975) Secretion of hypo-osmotic fluid by the iower Malpighian tubules of Rhodnius prollsus. J. esp. Biol. 62. 671-683. Nijhout H. F. (1981) Physiological control of molting in insects. .4m. Zool. 21, 631-640. Osborne M. P. and Finlayson L. H. (1962) The structure and topography of stretch receptors in representative orders of &cts. Q. JI Microsc. Sci. 103, 277-242. Rvckman R. E. and Rvckman A. E. ( 1967) Eoizootiologv of Trypanosoma &I in southwestern North America. Part X: The biosystematics of Dipetulogusrcr musimus in Mexico. J. med. En/. 4, 180-188. Van Der Kloot W. G. (1960) Neurosecretion in Insects. .% Rev. &I. 5, 35-52. Van Der Kloot W. G. (1961) Insect metamorphosis and Its endocrine control. Am. Zool. I. 3--9. Wigglesworth V. B. (1934) The physiology of ecdysis in Rhodnius proli.ws (Hemiptera). II. Factors controlling moulting and ‘metamorphosis’. Q, .I/. Mirmwi. Sci. 77. 19 l-222. Wigglesworth V. B. (lY36) The function of the corpus allatum in the growth and reproduction of Rhorlnius proli.ms (Hemiptera). Q. JI. Microsr. SC,;. 79, Yl- I2 I. Wigglesworth V. B. ( 1970) fnsecr Hornlonrs. I59 pp. Freeman. San Francisco.