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Quantitative studies on the activity of the corpora allata in adult male Locusta and Schistocerca
2. Insect Physiol., 1973, Vol. 19, pp. 2459 to 2467. Pergamon Press. Printed in Great Britain
QUANTITATIVE STUDIES ON THE ACTIVITY OF THE CORPORA ALLATA IN ADULT MALE LOCUSTA AND
SCHISTOCERCA R. A. JOHNSON1
and L. HILL2
lDepartment of Zoology, Imperial College, London; and aDepartment of Zoology, The University, Sheffield, England (Received 1 June 1973) Abstract-Juvenile hormone has been detected in the haemolymph and corpora allata of adult male Locusta and the haemolymph of adult male Schistocwa by a modified Galleria bioassay. The hormone was readily detected in the haemolymph of insects immediately after the final ecdysis, but then became difficult to detect until 2 days prior to the onset of sexual maturation. In sexually mature insects the titre of juvenile hormone was maintained at a constant level. The corpora allata of adult male Locusta increased in size throughout adult life. The juvenile hormone content of the corpora allata was low during the period of somatic growth, but increased at the onset of sexual maturation. Sectioning of the nervi corporis allati I in insects immediately after the final ecdysis prevented the normal increase in size of the corpora allata, but did not render them inactive since juvenile hormone was detected in the haemolymph after the operation. The half life of juvenile hormone in the haemolymph of allatectomized adult male Locusta was 1 to 2 hr. INTRODUCTION THE CORPORAALLATAof the adult male locust have been implicated in the control of a number of physiological processes. If the corpora allata are removed from adult male Locusta shortly after the final ecdysis, the accessory glands fail to develop, there is hypertrophy of the fat body (STRONG, 1968), mating behaviour is less intense (WAJC and PENER,1969), flight activity decreases(W~c~ and PENER,1971), and the life span of the insect is increased (WAJC and PENER,1969; PENER,1970). Whether these various effects are all the direct result of a lack of juvenile hormone is unknown. In studies on the physiological effects of juvenile hormone it is clearly important to know when the corpora allata release juvenile hormone. Yet until recently most measurements of corpus allatum activity have been indirect, using criteria such as gland size and gland histology. This type of measurement does not necessarily give a true indication of the activity of the corpora allata (JOHNSONand HILL, 1973). In this study the presence of juvenile hormone in the haemolymph and corpora allata of adult male Locustu and the haemolymph of adult male Schistocemz has been detected by a modified Galleria bioassay. The results obtained give a clear indication of the times when the .corpora allata release juvenile hormone into the
2459
2460
R. A. JOHNSON ANDL. HILL
haemolymph of the adult male locust, and provide some data on the control of corpus allatum activity and the half life of juvenile hormone in the haemolymph. MATERIALS
AND METHODS
Adult males of the African migratory locust, Locusta migratoria migratorioides, and the desert locust, Schistocerca gregaria, were taken from colonies reared under crowded conditions at a temperature of 30°C with a 12 hr photoperiod. A constant diet of fresh lettuce and bran was provided daily. The details of the modified Galleria bioassay for juvenile hormone, the methods for allatectomy, the measurement of somatic growth, haemolymph volume, and fresh corpus allatum volume have been previously described (JOHNSONand HILL, 1973). The protein content of five pairs of homogenized corpora allata was measured by the method of LAYNE (1957) using a bovine serum albumin standard. The nervi corporis allati I (NCAI) were sectioned as described by STAAL (1961). RESULTS Growth, haemolymph. volume, and changes in corpus allatum volumes The changes in the somatic dry weight (the total dry weight minus the weight of the fat body), the haemolymph volume, the volumes of the corpora allata, and the protein content of the corpora allata of adult male Locusta are shown in Table 1. The somatic dry weight increased during the first 10 days of adult life and then The haemolymph volume showed similar changes. The remained constant. corpora allata continued to increase in size throughout adult life, and this was reflected in increased protein content. The most rapid increase in the size of the corpora allata occurred during the period of somatic growth. The changes in the volumes of the corpora allata of Schistocerca are shown in Table 1. The corpora allata increased in volume during the period of somatic growth but then remained of constant volume. The corpora allata of Locusta are much larger than those of Schistocerca, but this is not apparent from Table 1, as the method for measuring fresh corpus allatum volumes gives only arbitrary size units, the starting point of these units depending upon initial gland size (STAAL, 1961). Juvenile hormone in the haemolynzph The results of the bioassay of haemolymph extracts for juvenile hormone activity in adult male Locusta are shown in Fig. 1. Two types of results are shown, the assay of the unpurified total haemolymph lipid extract, and the assay of the juvenile hormone fraction isolated by thin-layer chromatography. The purification of the total lipid extract clearly increased the sensitivity of the assay. Juvenile hormone was readily detected in the haemolymph of adult male Locusta immediately after the final ecdysis, but was then difficult to detect until 2 days before the In the sexually mature insect juvenile hormone was onset of sexual maturity. present in the haemolymph at a constant high’level. The results of the bioassay of extracts of haemolymph from adult male Schistocwca are shown in Fig. 2. These
229.7 * 15.4 268.9 + 16.9 370.2 k10.4 467.8 + 12.2 466.0 f 24.9 549.4 + 20.7 574.3 rt:34.0 595.5 + 11.4 544.0 * 35.9 566.2 f 14.1 494.2 + 16.3 521.l + 20.7 489.7 zk12.4
Age (days)
0 2 4 6 8 10 12 14 16 18 20 30 40 204.5 AZ21.9 241.3 I!I27.6 271.1 k 15.4 299.2 + 21*l 327.9 + 31.4 334.4 + 26.5 347.7 + 39.8 298.7 * 20.1 301.6 + 17.9 279.4 rf:19.1 290.7 + 23.1 267.1 rt 29.4 281.3 ?I 19.3
Haemolymph volume (~1 f S.E.) 13.67 + 1.01 10.82 + 0.53 23.47 + 0.42 31.29 + 0.86 38.33 z!z0.27 48.43 rt:3.51 52.43 + 3.91 56.11 f 2.57 57.11 Ik2.11 58.81 * 4.90 60.69 + 2.81 69.47 rt 2.47 77.16 + 2.79
Volumes of Locusta corpora allata (planimeter units k S.E.)
51.2 54.1 58.3
46.4 -
42.3 -
35.1 -
24.7 -
16.2 -
Protein content of 10 corpora ahata (pg)
Each figure, except the values for protein content of the corpora allata, is the mean of five observations.
Somatic dry weight (mg + S.E.)
15.65 + 0.96 19.59 * 0.50 21.71 rt 0.57 24.42 L!I1a09 27.95 z!I0.94 32.66 + 1a66 31.79 + 2.57 33.85 + 2.56 32.27 f 0.94 30.47 * I.29 30.55 * 1.11 30.34 * I.94 29.95 rt 1.45
Volumes of Schistocerca corpora allata (planimeter units If:SE.)
TABLE ~-THE CHANGES IN THE SOMATICDRY WEIGHT,HAEMOLYMPHVOLUMES, THE VOLUMES OF THE CORPORAALLATA,AND THE PROTEIN CONTENT0F THE CORPORA ALLAT. IN ADULT MALE Locusta, AND THE VOLUMESOF THE CORPORAALLATA IN ADULT MALE Schistocerca
R.A.
2462
JOHNSONAND L.HILL
results are identical with those in adult male Locusta, except that the onset of sexual maturation and the appearance of juvenile hormone in the haemolymph occurred 2 days later. Total
q
0
2
haemoiymph
Isolated
4
6
lipid
iuvenile
hormone
a
10
Age
(daus)
4
16
FIG. 1. The percentage response of Galleria pupae to the application of juvenile hormone extracts of haemolymph from adult male Locusta. The effects of a total haemolymph lipid extract and an extract containing juvenile hormone isolated from the haemolymph iipids are shown. The small black bars indicate 100 per cent negative results.
nnnn 2
4
1.
6
810
I2
Age
I4
16
IS
20
(days)
FIG. 2. The percentage response of Galleria pupae to the application of juvenile hormone extracts of haemolymph from adult male Schistocerca. The juvenile hormone was purified by separation from the haemolymph lipids.
ACTIVITY
juvenile
OF CORPORA ALLATA IN MALE LOCUSTA
AND SCHISTOCERCA
2463
hormone within the corpora allata
Ten pairs of corpora allata were removed from male Locusta at various stages in adult life. The fresh volumes of the corpora allata were measured and lipid extracts from these glands were assayed for juvenile hormone. The results are shown in Fig. 3. The corpora allata from insects immediately after the final ecdysis contained more juvenile hormone than did the corpora allata from 4 and 8 day old insects. With the onset of sexual maturity, the juvenile hormone content of the corpora allata increased but then remained constant throughout adult life, although the volumes of the corpora allata continued to increase. 100 t
FIG. 3. The percentage response of Galleria pupae to the application of purified extracts of juvenile hormone from the corpora allata and the volumes of the corpora allata of adult male Locusta.
Disappearance
of juvenile hormone from the haemolymph following
allatectomy
In a preliminary experiment 30 day old adult male Locusta were allatectomized and 500 ~1 samples of haemolymph were taken for bioassay 12 and 24 hr after the operation. A 20 per cent positive response was obtained 12 hr after the operation, but after 24 hr there was a 100 per cent negative response. A more detailed examination of the rate of disappearance of juvenile hormone from the haemolymph of 30 day old adult male Locusta during the first 12 hr after allatectomy was made using 100 ,ul samples of haemolymph. The results are shown
R. A. JOHNSONAND L. HILL
2464
in Table 2 and indicate that the half life of juvenile hormone in the haemolymph of adult male Locwta was of the order of 1 to 2 hr. TABLE ~---THE DISAPPRARANCE OF JUVENILE HORMONE FROMTHEHAEMOLYMPH OF 30 day old ADULTMALELOCUS&FOLLOWING ALLATECTOMY Time after the operation (hr)
Response in bioassay (%)
0 3 6
65 10 0
The effects of sectioning the NCAI on the volumes of the corpora allata and the presence of juvenile hormone in the haemo~~~~h Sectioning of the NCAI of adult male Locusta immediately after the final ecdysis prevented the normal increase in size of the corpora allata (Table 3). Juvenile hormone was, however, detected in the haemolymph of these animals, and the bioassay results did not differ from those of control animals (Table 3). TABLE3--THE EFFECTS OFSECTIONING THENRRVICORPORIS ALLATI1 IMMEDIATELY AFTERTHE FINAL
ECDYSIS
ON
THE
VOLUMES
OF
THE
HORMONE
CORPORA IN
THE
ALLATA
AND
THE
0 5 10 15 20
Controls
NCAI sectioned
13*02&0.79(S) 24.85 + 1.10 (5) 35.86 rf:1.13 (5) 51.55 f 3.46 (5) 63.55 k4.86 (5)
1468 21.06 25.01 25.36
f 0.23 + 1.07 & 2.79 + 1.46
OF
JUVENILE
Bioassay results (% response)
Volumes of the corpus allatum (planimeter units + S.E.)
Age (day4
PRESENCE
HAEMOLYMPH
(5) (5) (4) (4)
Comparison of means
Control
NCAI sectioned
P
20 10 10 100 100
10 10 100 100
The figures in parentheses refer to the number of observations in each group. TARLE ~-THE EFFECTS OF SECTIONING THENERVICORPORIS ALLATAI OF 30 day old ADULT MALE Locusta oiw THE VOLUMES OF THE CORPORA ALLATA AND THE PRESENCE OF JUVENILE HORMONE
IN
THE
HARMOLYMPH
Volumes of the corpus allatum (planimeter units + SE.)
30 day controls 33 day controls 33 day sham-operated 33 day NCAI section
91.50 94.76 90.72 49.63
+ + + f
4.71 5.34 5.11 2-72
(5) (5) (5) (5)
Bioassay results ( y0 response) 100 100 100 100
The figures in parentheses refer to the number of observations in each group.
ACTIVITYOF CORPORA ALLATA IN MALELOCUSTA ANDSCHISTOCERCA
2465
Sectioning of the NCAI of 30 day old adult male Locusta resulted in a significant decrease in the size of the corpora allata by 3 days after the operation, but no differences were detected between the juvenile hormone content of the haemolymph of operated and control animals (Table 4). DISCUSSION
Although the physiological importance of the corpora allata in adult male insects is well established (ENGELMANN, 1970), reliable data concerning their activity is sparse (MENDES, 1948; &BAS, 1957; SCHARRERand VON HARNACK, 1958; ODHIAMBO, 1966; JOLY, 1967). ODHIAMBO(1966) attempted to measure the activity of the corpora allata in adult male S. gregaria by the implantation of corpora allata into male Schistocewa allatectomized soon after the final ecdysis and noting the effects on accessory gland development and cuticle coloration in the host. This technique revealed that the glands were apparently inactive during the period of somatic growth, but became active towards the end of this period. The corpora allata of adult male Schistocerca increased in volume during the period of somatic growth, but following the onset of sexual maturation the volumes of the glands remained constant, and so a correlation was drawn between the volumes of the glands and their activity (ODHIAMBO, 1966). In this present study two criteria were used to estimate the activity of the corpora allata in adult male Locusta, namely changes in gland volumes and the presence of juvenile hormone in the haemolymph and corpora allata. Additionally, corpus allatum volumes and the presence of juvenile hormone in the haemolymph of adult male Schistocema were measured. A comparison of these two locust species reveals the inadequacy of using corpus allatum size as a criterion of gland activity. The corpora allata of adult male Locusta are larger than those of adult male Schistocerca, and the corpora allata of male Locusta continue to increase in size throughout adult life, whilst in adult male Schistocerca no increase in size occurs after somatic growth has finished. In both species, however, the bioassay results for the presence of juvenile hormone in the haemolymph are identical. The reason for the continued increase in size of the corpora allata in adult male Locusta is known. No mitosis occurs in the corpora allata during the adult instar of Locusta, but cytoplasmic vesicles appear in the glands at sexual maturity which increase in size and number with age (OZBAS, 1957; Johnson, unpublished observations). It has been suggested that lipid droplets in the corpora allata of Calliphora erythrocephala contain stored juvenile hormone (THOMSEN and THOMSEN, 1970). The vesicles in the corpora allata of Locusta might be a form of glandular storage, but the amount of juvenile hormone within the corpora allata does not increase following the onset of sexual maturation. This suggests that the corpora allata do not store appreciable quantities of juvenile hormone, at least in a physiologically active form. The bioassay results confirm the observations of ODHIAMBO (1966) that the corpora allata in locusts secrete large quantities of juvenile hormone towards the
2466
R. A. JOHNSON ANDL. HILL
end of the period of somatic growth. It is possible, however, that juvenile hormone is secreted at a very low level throughout the whole of the period of somatic growth. Juvenile hormone can be readily detected in the haemolymph during the day following the final ecdysis and is present in the haemolymph in smaller quantities throughout somatic growth. If the half life of the hormone in immature adult Locucta is similar to that in the sexually mature insect, then the corpora allata are clearly releasing small quantities of hormone throughout the period of somatic growth. When the corpora allata are removed from sexually mature adult male Locusta juvenile hormone rapidly disappears from the haemolymph. These experiments suggest a half life for Locusta juvenile hormone of 1 to 2 hr. Little is known of the half lives of insect hormones and so it is difficult to comment further on the physiological significance of this result. However, it is clear that in Locusta juvenile hormone must be rapidly utilized or broken down and to maintain a high level of hormone in the haemolymph the corpora allata must be constantly synthesizing and releasing the_ hormone. Juvenile hormone cannot be detected in locust faeces and so it is unlikely that the hormone is excreted unchanged. The regulation of corpus allatum activity is problematical, and it is likely that no single controlling mechanism can be applied to all insects (HIGHNAM, 1967 ; ENGELMANN,1970). The intermittent activity of the corpora allata in Locusta has been clearly demonstrated in this and an earlier paper (JOHNSONand HILL, 1973). The amount of juvenile hormone in the corpora allata of adult male Locusta rises sharply shortly before the onset of sexual maturation, concomitant with the appearance of juvenile hormone in the haemolymph. This suggests that in adult male Locusta the synthesis and release of juvenile hormone are initiated at the same time. The fact that the corpora allata are able to release juvenile hormone after section of the NCAI is difficult to reconcile with previous work on the control of corpus allatum activity. The C cells of the median neurosecretory cells of the pars intercerebralis have been implicated in the control of corpus allatum activity in Locusta (GIRARDIE, 1966, 1967; PENER et aZ., 1973, but clearly intact nervous connexions between the brain and the corpora allata are not necessary for juvenile hormone synthesis and release. The size of the corpora allata, however, is dependent on intact nervous connexions with the brain, although gland size is clearly shown by these experiments to be an unsatisfactory criterion of gland activity. However, the measurements of juvenile hormone in the haemolymph reported in this study are not strictly quantitative and so it is possible that the titre of juvenile hormone in the haemolymph is lower after nerve section, although still present in physiologically active quantities. It is also possible that the cut ends of the NCAI are able to release sufficient neurosecretory hormone into the haemolymph to activate the corpora allata. Acknowledgement-R. A. J. is grateful to the Science Research Council for the provision of a Studentship in the Department of Zoology, The University of Sheffield, where this work was performed.
ACTIVITYOF CORPORA ALLATAIN MALELOCUSTA
AND SCHISTOCERCA
2467
REFERENCES ENGELMANNF. (1970) The Physiology of Insect Reproduction. Pergamon Press, Oxford. GIRARDIEA. (1966) ContrBle de l’activite genitale chez Locusta migratoria. Mise en evidence d’un facteur gonadotrope et d’un facteur allatotrope dans la pars intercerebralis. Bull. Sot. zool. Fr. 91, 423-439. GIRARDIEA. (1967) Contrble neuro-hormonal de la m&amorphose et de la pigmentation chez Locusta migratoria cincerascens (Orthopdre). Bull. Biol. Fr. Belg. 101, 79-114. HIGHNAMK. C. (1967) Insect hormones. J. Endocr. 39, 123-150. JOHNSONR. A. and HILL L. (1973) The activity of the corpora allata in the fourth and fifth larval instars of the migratory locust. J. Insect Physiol. In press. JOLY P. (1967) Comparaison du volume et de I’activite physiologique des corpora allata de Locusta migratoria. Ann. Sot. ent. Fr. 3, 601-608. LAYNE E. (1957) Spectrophotometric and turbidometric methods for measuring proteins. Methods in Enzymology 3,448. Academic Press, New York. MENDESM. (1948) Histology of the corpora allata of Melanoplus di&ventialis. Biol. Bull., Woods Hole 94, 194-207. ODHIAMBOT. R. (1966) Morphometric changes and the hormonal activity of the corpus allatum in the adult male of the desert locust. J. Insect Physiol. 12, 655-664. OZBAS S. (1957) Morphological and histological studies on the corpora allata and cardiaca in Orthoptera. Comm. Fat. 5%. Ankara (C) 8, 19-43. PENERM. P. (1970) The corpus allatum in adult acridids: the interrelation of its function and possible correlations with the life cycle. Proc. Int. Study Conf. Current Future Problems Acridology, London, pp. 135-147. PFNERM. P., GIRARDIEA., and JOLYP. (1972) Neurosecretory and corpus allatum controlled effects on mating behavior and colour change in adult Locusta migratoria migratorioides males. Gen. camp. Endocr. 19, 494-508. SCHARRER B. and VON HARNACKM. (1958) Histophysiological studies on the corpus allatum of Leucophaea maderae-I. Normal life cycle in male and female adults. Biol. Bull., Woods Hole 115, 508-520. STAAL G. B. (1961) Studies on the physiology of phase induction in Locusta migratoria migratorioides R and F. Publ. Fonds Landb. Export Bur. No. 40. STRONGL. (1968) The effect of enforced locomotor activity on lipid content in allatectomized males of Locusta migratoria migratorioides. J. exp. Biol. 48, 625-630. THOMSENE. and THOMSENM. (1970) Fine structure of the corpus allatum of the female blow-fly Calliphora erythrocephala. Z. Zellforsch. micr. Anat. 110,40-60. WAJC E. and PENERM. P. (1969) The effect of the corpora allata on the mating behavior of the male migratory locust, Locusta migratoria migratorioides (R and F). Isr. 3. Zool. 18, 179-192. WAJC E. and PENERM. P. (1971) The effect of the corpora allata on flight activity of the male African migratory locust, Locusta migratoria migratorioides (R and F). Gen. camp. Endocr. 17, 327-333.
QUANTITATIVE STUDIES ON THE ACTIVITY OF THE CORPORA ALLATA IN ADULT MALE LOCUSTA AND
SCHISTOCERCA R. A. JOHNSON1
and L. HILL2
lDepartment of Zoology, Imperial College, London; and aDepartment of Zoology, The University, Sheffield, England (Received 1 June 1973) Abstract-Juvenile hormone has been detected in the haemolymph and corpora allata of adult male Locusta and the haemolymph of adult male Schistocwa by a modified Galleria bioassay. The hormone was readily detected in the haemolymph of insects immediately after the final ecdysis, but then became difficult to detect until 2 days prior to the onset of sexual maturation. In sexually mature insects the titre of juvenile hormone was maintained at a constant level. The corpora allata of adult male Locusta increased in size throughout adult life. The juvenile hormone content of the corpora allata was low during the period of somatic growth, but increased at the onset of sexual maturation. Sectioning of the nervi corporis allati I in insects immediately after the final ecdysis prevented the normal increase in size of the corpora allata, but did not render them inactive since juvenile hormone was detected in the haemolymph after the operation. The half life of juvenile hormone in the haemolymph of allatectomized adult male Locusta was 1 to 2 hr. INTRODUCTION THE CORPORAALLATAof the adult male locust have been implicated in the control of a number of physiological processes. If the corpora allata are removed from adult male Locusta shortly after the final ecdysis, the accessory glands fail to develop, there is hypertrophy of the fat body (STRONG, 1968), mating behaviour is less intense (WAJC and PENER,1969), flight activity decreases(W~c~ and PENER,1971), and the life span of the insect is increased (WAJC and PENER,1969; PENER,1970). Whether these various effects are all the direct result of a lack of juvenile hormone is unknown. In studies on the physiological effects of juvenile hormone it is clearly important to know when the corpora allata release juvenile hormone. Yet until recently most measurements of corpus allatum activity have been indirect, using criteria such as gland size and gland histology. This type of measurement does not necessarily give a true indication of the activity of the corpora allata (JOHNSONand HILL, 1973). In this study the presence of juvenile hormone in the haemolymph and corpora allata of adult male Locustu and the haemolymph of adult male Schistocemz has been detected by a modified Galleria bioassay. The results obtained give a clear indication of the times when the .corpora allata release juvenile hormone into the
2459
2460
R. A. JOHNSON ANDL. HILL
haemolymph of the adult male locust, and provide some data on the control of corpus allatum activity and the half life of juvenile hormone in the haemolymph. MATERIALS
AND METHODS
Adult males of the African migratory locust, Locusta migratoria migratorioides, and the desert locust, Schistocerca gregaria, were taken from colonies reared under crowded conditions at a temperature of 30°C with a 12 hr photoperiod. A constant diet of fresh lettuce and bran was provided daily. The details of the modified Galleria bioassay for juvenile hormone, the methods for allatectomy, the measurement of somatic growth, haemolymph volume, and fresh corpus allatum volume have been previously described (JOHNSONand HILL, 1973). The protein content of five pairs of homogenized corpora allata was measured by the method of LAYNE (1957) using a bovine serum albumin standard. The nervi corporis allati I (NCAI) were sectioned as described by STAAL (1961). RESULTS Growth, haemolymph. volume, and changes in corpus allatum volumes The changes in the somatic dry weight (the total dry weight minus the weight of the fat body), the haemolymph volume, the volumes of the corpora allata, and the protein content of the corpora allata of adult male Locusta are shown in Table 1. The somatic dry weight increased during the first 10 days of adult life and then The haemolymph volume showed similar changes. The remained constant. corpora allata continued to increase in size throughout adult life, and this was reflected in increased protein content. The most rapid increase in the size of the corpora allata occurred during the period of somatic growth. The changes in the volumes of the corpora allata of Schistocerca are shown in Table 1. The corpora allata increased in volume during the period of somatic growth but then remained of constant volume. The corpora allata of Locusta are much larger than those of Schistocerca, but this is not apparent from Table 1, as the method for measuring fresh corpus allatum volumes gives only arbitrary size units, the starting point of these units depending upon initial gland size (STAAL, 1961). Juvenile hormone in the haemolynzph The results of the bioassay of haemolymph extracts for juvenile hormone activity in adult male Locusta are shown in Fig. 1. Two types of results are shown, the assay of the unpurified total haemolymph lipid extract, and the assay of the juvenile hormone fraction isolated by thin-layer chromatography. The purification of the total lipid extract clearly increased the sensitivity of the assay. Juvenile hormone was readily detected in the haemolymph of adult male Locusta immediately after the final ecdysis, but was then difficult to detect until 2 days before the In the sexually mature insect juvenile hormone was onset of sexual maturity. present in the haemolymph at a constant high’level. The results of the bioassay of extracts of haemolymph from adult male Schistocwca are shown in Fig. 2. These
229.7 * 15.4 268.9 + 16.9 370.2 k10.4 467.8 + 12.2 466.0 f 24.9 549.4 + 20.7 574.3 rt:34.0 595.5 + 11.4 544.0 * 35.9 566.2 f 14.1 494.2 + 16.3 521.l + 20.7 489.7 zk12.4
Age (days)
0 2 4 6 8 10 12 14 16 18 20 30 40 204.5 AZ21.9 241.3 I!I27.6 271.1 k 15.4 299.2 + 21*l 327.9 + 31.4 334.4 + 26.5 347.7 + 39.8 298.7 * 20.1 301.6 + 17.9 279.4 rf:19.1 290.7 + 23.1 267.1 rt 29.4 281.3 ?I 19.3
Haemolymph volume (~1 f S.E.) 13.67 + 1.01 10.82 + 0.53 23.47 + 0.42 31.29 + 0.86 38.33 z!z0.27 48.43 rt:3.51 52.43 + 3.91 56.11 f 2.57 57.11 Ik2.11 58.81 * 4.90 60.69 + 2.81 69.47 rt 2.47 77.16 + 2.79
Volumes of Locusta corpora allata (planimeter units k S.E.)
51.2 54.1 58.3
46.4 -
42.3 -
35.1 -
24.7 -
16.2 -
Protein content of 10 corpora ahata (pg)
Each figure, except the values for protein content of the corpora allata, is the mean of five observations.
Somatic dry weight (mg + S.E.)
15.65 + 0.96 19.59 * 0.50 21.71 rt 0.57 24.42 L!I1a09 27.95 z!I0.94 32.66 + 1a66 31.79 + 2.57 33.85 + 2.56 32.27 f 0.94 30.47 * I.29 30.55 * 1.11 30.34 * I.94 29.95 rt 1.45
Volumes of Schistocerca corpora allata (planimeter units If:SE.)
TABLE ~-THE CHANGES IN THE SOMATICDRY WEIGHT,HAEMOLYMPHVOLUMES, THE VOLUMES OF THE CORPORAALLATA,AND THE PROTEIN CONTENT0F THE CORPORA ALLAT. IN ADULT MALE Locusta, AND THE VOLUMESOF THE CORPORAALLATA IN ADULT MALE Schistocerca
R.A.
2462
JOHNSONAND L.HILL
results are identical with those in adult male Locusta, except that the onset of sexual maturation and the appearance of juvenile hormone in the haemolymph occurred 2 days later. Total
q
0
2
haemoiymph
Isolated
4
6
lipid
iuvenile
hormone
a
10
Age
(daus)
4
16
FIG. 1. The percentage response of Galleria pupae to the application of juvenile hormone extracts of haemolymph from adult male Locusta. The effects of a total haemolymph lipid extract and an extract containing juvenile hormone isolated from the haemolymph iipids are shown. The small black bars indicate 100 per cent negative results.
nnnn 2
4
1.
6
810
I2
Age
I4
16
IS
20
(days)
FIG. 2. The percentage response of Galleria pupae to the application of juvenile hormone extracts of haemolymph from adult male Schistocerca. The juvenile hormone was purified by separation from the haemolymph lipids.
ACTIVITY
juvenile
OF CORPORA ALLATA IN MALE LOCUSTA
AND SCHISTOCERCA
2463
hormone within the corpora allata
Ten pairs of corpora allata were removed from male Locusta at various stages in adult life. The fresh volumes of the corpora allata were measured and lipid extracts from these glands were assayed for juvenile hormone. The results are shown in Fig. 3. The corpora allata from insects immediately after the final ecdysis contained more juvenile hormone than did the corpora allata from 4 and 8 day old insects. With the onset of sexual maturity, the juvenile hormone content of the corpora allata increased but then remained constant throughout adult life, although the volumes of the corpora allata continued to increase. 100 t
FIG. 3. The percentage response of Galleria pupae to the application of purified extracts of juvenile hormone from the corpora allata and the volumes of the corpora allata of adult male Locusta.
Disappearance
of juvenile hormone from the haemolymph following
allatectomy
In a preliminary experiment 30 day old adult male Locusta were allatectomized and 500 ~1 samples of haemolymph were taken for bioassay 12 and 24 hr after the operation. A 20 per cent positive response was obtained 12 hr after the operation, but after 24 hr there was a 100 per cent negative response. A more detailed examination of the rate of disappearance of juvenile hormone from the haemolymph of 30 day old adult male Locusta during the first 12 hr after allatectomy was made using 100 ,ul samples of haemolymph. The results are shown
R. A. JOHNSONAND L. HILL
2464
in Table 2 and indicate that the half life of juvenile hormone in the haemolymph of adult male Locwta was of the order of 1 to 2 hr. TABLE ~---THE DISAPPRARANCE OF JUVENILE HORMONE FROMTHEHAEMOLYMPH OF 30 day old ADULTMALELOCUS&FOLLOWING ALLATECTOMY Time after the operation (hr)
Response in bioassay (%)
0 3 6
65 10 0
The effects of sectioning the NCAI on the volumes of the corpora allata and the presence of juvenile hormone in the haemo~~~~h Sectioning of the NCAI of adult male Locusta immediately after the final ecdysis prevented the normal increase in size of the corpora allata (Table 3). Juvenile hormone was, however, detected in the haemolymph of these animals, and the bioassay results did not differ from those of control animals (Table 3). TABLE3--THE EFFECTS OFSECTIONING THENRRVICORPORIS ALLATI1 IMMEDIATELY AFTERTHE FINAL
ECDYSIS
ON
THE
VOLUMES
OF
THE
HORMONE
CORPORA IN
THE
ALLATA
AND
THE
0 5 10 15 20
Controls
NCAI sectioned
13*02&0.79(S) 24.85 + 1.10 (5) 35.86 rf:1.13 (5) 51.55 f 3.46 (5) 63.55 k4.86 (5)
1468 21.06 25.01 25.36
f 0.23 + 1.07 & 2.79 + 1.46
OF
JUVENILE
Bioassay results (% response)
Volumes of the corpus allatum (planimeter units + S.E.)
Age (day4
PRESENCE
HAEMOLYMPH
(5) (5) (4) (4)
Comparison of means
Control
NCAI sectioned
P
20 10 10 100 100
10 10 100 100
The figures in parentheses refer to the number of observations in each group. TARLE ~-THE EFFECTS OF SECTIONING THENERVICORPORIS ALLATAI OF 30 day old ADULT MALE Locusta oiw THE VOLUMES OF THE CORPORA ALLATA AND THE PRESENCE OF JUVENILE HORMONE
IN
THE
HARMOLYMPH
Volumes of the corpus allatum (planimeter units + SE.)
30 day controls 33 day controls 33 day sham-operated 33 day NCAI section
91.50 94.76 90.72 49.63
+ + + f
4.71 5.34 5.11 2-72
(5) (5) (5) (5)
Bioassay results ( y0 response) 100 100 100 100
The figures in parentheses refer to the number of observations in each group.
ACTIVITYOF CORPORA ALLATA IN MALELOCUSTA ANDSCHISTOCERCA
2465
Sectioning of the NCAI of 30 day old adult male Locusta resulted in a significant decrease in the size of the corpora allata by 3 days after the operation, but no differences were detected between the juvenile hormone content of the haemolymph of operated and control animals (Table 4). DISCUSSION
Although the physiological importance of the corpora allata in adult male insects is well established (ENGELMANN, 1970), reliable data concerning their activity is sparse (MENDES, 1948; &BAS, 1957; SCHARRERand VON HARNACK, 1958; ODHIAMBO, 1966; JOLY, 1967). ODHIAMBO(1966) attempted to measure the activity of the corpora allata in adult male S. gregaria by the implantation of corpora allata into male Schistocewa allatectomized soon after the final ecdysis and noting the effects on accessory gland development and cuticle coloration in the host. This technique revealed that the glands were apparently inactive during the period of somatic growth, but became active towards the end of this period. The corpora allata of adult male Schistocerca increased in volume during the period of somatic growth, but following the onset of sexual maturation the volumes of the glands remained constant, and so a correlation was drawn between the volumes of the glands and their activity (ODHIAMBO, 1966). In this present study two criteria were used to estimate the activity of the corpora allata in adult male Locusta, namely changes in gland volumes and the presence of juvenile hormone in the haemolymph and corpora allata. Additionally, corpus allatum volumes and the presence of juvenile hormone in the haemolymph of adult male Schistocema were measured. A comparison of these two locust species reveals the inadequacy of using corpus allatum size as a criterion of gland activity. The corpora allata of adult male Locusta are larger than those of adult male Schistocerca, and the corpora allata of male Locusta continue to increase in size throughout adult life, whilst in adult male Schistocerca no increase in size occurs after somatic growth has finished. In both species, however, the bioassay results for the presence of juvenile hormone in the haemolymph are identical. The reason for the continued increase in size of the corpora allata in adult male Locusta is known. No mitosis occurs in the corpora allata during the adult instar of Locusta, but cytoplasmic vesicles appear in the glands at sexual maturity which increase in size and number with age (OZBAS, 1957; Johnson, unpublished observations). It has been suggested that lipid droplets in the corpora allata of Calliphora erythrocephala contain stored juvenile hormone (THOMSEN and THOMSEN, 1970). The vesicles in the corpora allata of Locusta might be a form of glandular storage, but the amount of juvenile hormone within the corpora allata does not increase following the onset of sexual maturation. This suggests that the corpora allata do not store appreciable quantities of juvenile hormone, at least in a physiologically active form. The bioassay results confirm the observations of ODHIAMBO (1966) that the corpora allata in locusts secrete large quantities of juvenile hormone towards the
2466
R. A. JOHNSON ANDL. HILL
end of the period of somatic growth. It is possible, however, that juvenile hormone is secreted at a very low level throughout the whole of the period of somatic growth. Juvenile hormone can be readily detected in the haemolymph during the day following the final ecdysis and is present in the haemolymph in smaller quantities throughout somatic growth. If the half life of the hormone in immature adult Locucta is similar to that in the sexually mature insect, then the corpora allata are clearly releasing small quantities of hormone throughout the period of somatic growth. When the corpora allata are removed from sexually mature adult male Locusta juvenile hormone rapidly disappears from the haemolymph. These experiments suggest a half life for Locusta juvenile hormone of 1 to 2 hr. Little is known of the half lives of insect hormones and so it is difficult to comment further on the physiological significance of this result. However, it is clear that in Locusta juvenile hormone must be rapidly utilized or broken down and to maintain a high level of hormone in the haemolymph the corpora allata must be constantly synthesizing and releasing the_ hormone. Juvenile hormone cannot be detected in locust faeces and so it is unlikely that the hormone is excreted unchanged. The regulation of corpus allatum activity is problematical, and it is likely that no single controlling mechanism can be applied to all insects (HIGHNAM, 1967 ; ENGELMANN,1970). The intermittent activity of the corpora allata in Locusta has been clearly demonstrated in this and an earlier paper (JOHNSONand HILL, 1973). The amount of juvenile hormone in the corpora allata of adult male Locusta rises sharply shortly before the onset of sexual maturation, concomitant with the appearance of juvenile hormone in the haemolymph. This suggests that in adult male Locusta the synthesis and release of juvenile hormone are initiated at the same time. The fact that the corpora allata are able to release juvenile hormone after section of the NCAI is difficult to reconcile with previous work on the control of corpus allatum activity. The C cells of the median neurosecretory cells of the pars intercerebralis have been implicated in the control of corpus allatum activity in Locusta (GIRARDIE, 1966, 1967; PENER et aZ., 1973, but clearly intact nervous connexions between the brain and the corpora allata are not necessary for juvenile hormone synthesis and release. The size of the corpora allata, however, is dependent on intact nervous connexions with the brain, although gland size is clearly shown by these experiments to be an unsatisfactory criterion of gland activity. However, the measurements of juvenile hormone in the haemolymph reported in this study are not strictly quantitative and so it is possible that the titre of juvenile hormone in the haemolymph is lower after nerve section, although still present in physiologically active quantities. It is also possible that the cut ends of the NCAI are able to release sufficient neurosecretory hormone into the haemolymph to activate the corpora allata. Acknowledgement-R. A. J. is grateful to the Science Research Council for the provision of a Studentship in the Department of Zoology, The University of Sheffield, where this work was performed.
ACTIVITYOF CORPORA ALLATAIN MALELOCUSTA
AND SCHISTOCERCA
2467
REFERENCES ENGELMANNF. (1970) The Physiology of Insect Reproduction. Pergamon Press, Oxford. GIRARDIEA. (1966) ContrBle de l’activite genitale chez Locusta migratoria. Mise en evidence d’un facteur gonadotrope et d’un facteur allatotrope dans la pars intercerebralis. Bull. Sot. zool. Fr. 91, 423-439. GIRARDIEA. (1967) Contrble neuro-hormonal de la m&amorphose et de la pigmentation chez Locusta migratoria cincerascens (Orthopdre). Bull. Biol. Fr. Belg. 101, 79-114. HIGHNAMK. C. (1967) Insect hormones. J. Endocr. 39, 123-150. JOHNSONR. A. and HILL L. (1973) The activity of the corpora allata in the fourth and fifth larval instars of the migratory locust. J. Insect Physiol. In press. JOLY P. (1967) Comparaison du volume et de I’activite physiologique des corpora allata de Locusta migratoria. Ann. Sot. ent. Fr. 3, 601-608. LAYNE E. (1957) Spectrophotometric and turbidometric methods for measuring proteins. Methods in Enzymology 3,448. Academic Press, New York. MENDESM. (1948) Histology of the corpora allata of Melanoplus di&ventialis. Biol. Bull., Woods Hole 94, 194-207. ODHIAMBOT. R. (1966) Morphometric changes and the hormonal activity of the corpus allatum in the adult male of the desert locust. J. Insect Physiol. 12, 655-664. OZBAS S. (1957) Morphological and histological studies on the corpora allata and cardiaca in Orthoptera. Comm. Fat. 5%. Ankara (C) 8, 19-43. PENERM. P. (1970) The corpus allatum in adult acridids: the interrelation of its function and possible correlations with the life cycle. Proc. Int. Study Conf. Current Future Problems Acridology, London, pp. 135-147. PFNERM. P., GIRARDIEA., and JOLYP. (1972) Neurosecretory and corpus allatum controlled effects on mating behavior and colour change in adult Locusta migratoria migratorioides males. Gen. camp. Endocr. 19, 494-508. SCHARRER B. and VON HARNACKM. (1958) Histophysiological studies on the corpus allatum of Leucophaea maderae-I. Normal life cycle in male and female adults. Biol. Bull., Woods Hole 115, 508-520. STAAL G. B. (1961) Studies on the physiology of phase induction in Locusta migratoria migratorioides R and F. Publ. Fonds Landb. Export Bur. No. 40. STRONGL. (1968) The effect of enforced locomotor activity on lipid content in allatectomized males of Locusta migratoria migratorioides. J. exp. Biol. 48, 625-630. THOMSENE. and THOMSENM. (1970) Fine structure of the corpus allatum of the female blow-fly Calliphora erythrocephala. Z. Zellforsch. micr. Anat. 110,40-60. WAJC E. and PENERM. P. (1969) The effect of the corpora allata on the mating behavior of the male migratory locust, Locusta migratoria migratorioides (R and F). Isr. 3. Zool. 18, 179-192. WAJC E. and PENERM. P. (1971) The effect of the corpora allata on flight activity of the male African migratory locust, Locusta migratoria migratorioides (R and F). Gen. camp. Endocr. 17, 327-333.