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Haemolymph ecdysteroid titres during larval-adult development in Rhodnius prolixus: Correlations with moulting hormone action and brain neurosecretory cell activity
.I. Insect Phpiof. Vol. 28, No. 6, pp. 519-525, 1982 Printed in Great Brifoin.
~22-1910/82/o60s19-07~3~~/0 0 1982 Pergamon Press Od
HAEMOLYMPH ECDYSTEROID TITRES LARVAL-ADULT DEVELOPMENT
DURING
IN
R~O~~~US ~RO~~X~S~ CORRELATIONS WITH MOULTING HORMONE ACTION AND BRAIN NEUROSECRETORY CELL ACTIVITY C. 6. H. STEEL*, W. E. BOLLENBACHER~, S. L. SMITH$and L. I. GI~BERH *Department of Biology, York University, Downsview, Ontario M3J lP3, Canada; fDepartment of Zoology, University of North Carolina, Chapel Hill, NC 27.514, U.S.A, and ~~part~nt of Biological Sciences, Bo~lj~g Green State University, Bowling Green. OH 43403. U.S.A. (Received 23 October 1981) Abstract-A haemolymph ecdysteroid titre of the fifth (last)-larval instar of the hemipteran, Rhodnius prolixus has been determined by radioimmunoassay. During the last-larval stadium the ecdysteroid titre increases from a negligible level in the unfed insect to a detectable level within minutes following a blood meal. The titre reaches a plateau of + 50-70 ng/ml at 3-4 hr and this level is maintained until day 5-6, the time of the head-critical period in Rkodnjus. At the head-critical period the titre begins to increase again, this time dramatically, reaching a peak of -3500 ng/ml at day 13. From day 14 to ecdysis (day 21) the titre declines to a low level, _ 30 r&ml. Basal levels of ecdysteroids, + 15 ng/mI, were detectable in young adult males and females. A survey of haemoiymph volumes during the last-larval instar indicates that the changes in the ecdysteroid titre reflect changes in the rates of ecdysteroid synthesis, and not changes in haemolymph volume. Excretion of ecdysteroids varies systematically during the instar, suggesting that control of ecdysteroid excretion may be important in regulation of the haemolymph titre. Qualitative analysis of the haemolymph ecdysteroid,RIA activity revealed the presence of only ecdysone and 20-hydrox~e~dysone. For the large peak preceding larval-adult ecdysis, 2~hydroxy-ecdysone was the predominant hormone. These rest&s indicate that there may be two periods of release of prothoracicotropic hormone (PTTH) from the brain in Rk~n~us, one immediately following the biood meal and the second on day 5 or 6. The significance of these times of PTTH release is discussed in relation to classical evidence of the timing of mouiting hormone action, the response of target tissues, and with more recent findings on the timing of release of neurosecretory material from the brain of Rkodnius during moulting. Key Word Index: Ecdysteroids, moulting, neurosecretion. prothoracicotropic hormone, Rkodnius
with the pioneering studies of Wigglesworth, research on the blood-sucking bug, Rho&us prolixus, has contributed substantially to our understanding of the endocrine regulation of growth and development in insects. Early observations that one cycle of moulting was initiated by a blood meal revealed that Rhodnius was an ideal insect for investigating the physiology of moulting on a precise timetable (URIBE, 1927; BUXTON,1930). Its usefulness as an experimental animal was enhanced by the fact that this bug could be surgically manipulated (e.g. decapitation, parabiosis) without the dangers of interference with continued feeding. The numerous publications on Rho~nius by Wi~lesworth (see LOCKEand SMITH 1980) provided considerable insight into the diverse tissue changes occurring during insect growth and development, and the endocrine regulation of these changes by hormones from the brain, corpora allata and prothoracic glands. It is ironic, considering the role of Rhodnius in establishing the dogma of the endo~rinolo~~l control of insect moulting and metamorphosis, that the titres of the hormones involved have not yet been determined. ~O~NC~G
The present paper reports an analysis of the ecdysteroid titre for the fifth (last)-larval instar of Rhodnius. This study details both quantitative and qualitative aspects of the titre. The significance of the titre is discussed in relation to classical studies on the secretion and effects of the moulting hormone on various tissues (see WIGGLESWORTH,1957, 1964), together with more recent studies on the role of neuros~retion from the brain in the control of moulting in Rhodnius (STEEL,1978. 1982). MATERIALS
AND MXTI-IODS
Animals ~~~~iu~ proiixus, derived from the culture used by Wigglesworth, were raised at 28 t_ 1°C an$ about 70% r.h. Fifth-instar larvae were timed from the blood meal (day 0). At this temperature adult ecdysis occurs 5 21 days after the blood meal. Wigglesworth’s early work employed Rhodn~us reared at 24°C with the fifth-larval stadium lasting -26 days (WIGGLESWORTH, 1934). To facilitate comparisons betweeni these investigations and the earlier studies of Wiggles:, worth, the timing given at 24°C has been adjusted, t&
519
520
C. G. H.
28°C in proportion to the interval from feeding to ecdysis at the two temperatures. With this procedure the assumption is made that all developmental events in the fifth-instar larvae are equally temperature sensitive. Although the adjusted times are regarded as approximate, this method is accurate for all events whose timing is known at both temperatures and indicates that the overall moulting cycle has a Qlo close to 2. Co~leetion of huemo~ymph, urine u~~eces
Haemolymph was expressed from the animals through a tibia1 cut and collected with a calibrated glass capillary. With moderate finger pressure on the animal, haemolymph was collected until it ceased to emerge in a steady stream. The volume of haemolymph collected in this manner is referred to as the expressible haemolymph volume and is used as a relative index of the haemolymph volume in an animal. Actual haemolymph volumes have been determined only for the early portion of the last-larval stadium (MADDRELL and GARDINER, 1980) in which the expressible haemolymph volume is a fairly constant 45% of the total. A similar proportion is expressible in the adult (GRINGORTENand FRIEND, 1979). This volume is therefore useful as an index of trends, although not of absolute volumes. During the profuse diuresis which occurs during the first few hours following feeding, fifth-instar Rhodnius excrete a watery urine equivalent to about three times their unfed weight (MADDRELL,1964). The total volume of urine produced by individual animals during diuresis was collected on filter paper and the volume of the urine determined from the loss in v$&ht of the animal. The filter paper was permitted to dry, extracted with methanol, and the methanolic extract assayed for ecdysteroids. Following diuresis, evacuation of the rectum in Rhodnius was carried out by handling the animals. Rectal contents were collected for extraction and assay of ecdysteroids.
STEELet aI.
previously (SMITHet al., 1979). Final identification of the ecdysteroids was by mass spectrometry.
RESULTS Haemolymph ecdysteroid titre
The taking of a blood meal by the larvae of Rhodof arrested development and the initiation of a moulting cycle. At feeding, the fifth&star larva ingests blood equal to * 10 times its weight in - 10 min. The amount of ecdysteraid RIA activity in the haemolymph of an unfed animal is negligible, but within minutes after the larva begins to feed the ecdysteroid titre begins to increase. The titre increases linearly and rapidly for 3-4 hr, at which time the titre is 5 70 ng/ml haemolymph (Fig. 1). This significant (P < 0.01) increase in the ecdysteroid titre between 0 and 3 hr may indicate the time of the first release of PTTH for larval-adult development in Rhodnius (see Discussion). From 4 to 24 hr post-feeding the titre decreases gradually to a level of -50 ng/ml haemolymph. This apparent decrease between 4 and 24 hr is not statistically significant (P > 0.5). From day 1 of the last instar to day 5 the ecdysteroid titre remains constant at w 50 ng/ml, but between days 5 and 7 (during the time of the classical head critical period} the titre begins to increase again, reaching its highest level (about 3~n~rnl~ during the 21-day stadium on day 13 (Fig. 2). This latter increase in the titre is probably in response to the release of PTTH at this head-critical period (It should be noted that further analyses at day 13-14 have resulted in mean values of 78OOng/ml). The titre remains relatively unchanged during days 13 and 14 and then drops dramatically by day 16 (- 800ng/ml) reaching a low level of 30ng/ml on day 20. Up to 3 days post-ecdysis, adult males and females exhibit a nius results in the termination
Ecdysteroid titre and chemistry
Ecdysteroids were extracted from haemolymph, urine and faeces by homogenization in methanol. After centrifugation at 8000 g for 10 min, the ecdysteroids in the methanolic extracts were quantified by radioimmunoassay (RIA) with D-10 antiserum which binds ecdysone and 20-hydroxy-ecdysone equally (BORSTand O’CONNOR,1974; GILBERT et al., 1977). RIA activity is expressed in 20-hydroxy-ecdysone equivalents since this ecdysteroid was used as the standard. The quantitative accuracy of the RIA was ascertained by the addition of 20-hydroxy-ecdysone as an internal standard as well as by purification of the ecdysteroids. All samples were assayed in duplicate, usually at 3-5 different concentrations. The labelled ligand for the RIA was [23,24-3H]-ecdysone ( - 57 Ci/mmol). Statistical analyses of the titre data were by Student’s t-test and non-parametric analysis of variance (KRUSKALand WALLI~,1952). Chemical analysis of the haemol~ph ecdysteroids was carried out by thin layer chromato~aphy (TLC), analytical normal and reverse phase high pressure liquid chromatography (HPLC) and RIA as described
Fig. 1. H~molymph ecdysteroid titres shortly after the initiation of larval-adult development at feeding. Ecdysteroids are undete~tabie prior to feeding but have increased to significant levels within 30 min. The level reached at 3-4 br is maintained for 5 days.
tines in Rhodnius
Ecdysteroid
0 J-0
J-4
_.-.-.
521
\
w* /6
B
I’0
l-2
1’4
Days after feeding
lb
‘-._
l’s
.-.
lo
v3
+ ecdysis
Fig. 2. Haemolymph ecdysteroid titres during larval-adult development. Standard errors for some of the samples are smaller than the point symbols. Time axis is interrupted at ecdysis and the samples taken thereafter are grouped by time since ecdysis.
marginally (Fig. 2). Haemolymph
detectable
ecdysteroid
titre of _ 15 ng/ml
volume
Due to the nature of the induction of the moult cycle in Rhodnius, the distinct possibility exists that the haemoiymph volume in a larva may vary dramatically following the blood meal, and this may give rise to an apparent increase or decrease in the ecdysteroid titre depending on whether haemolymph volume increases or decreases. To examine the possibility that fluctuations in the haemolymph volume may result in
Fig. 3. Expressible haemolymph these are both qualitatively
an apparent change in the haemolymph ecdysteroid titre, the expressible haemolymph volumes during the last-larval stadium have been determined and these volumes compared to the ecdysteroid titre during this period. During the last-larval stadium of Rhodnius, the expressible haemolymph volume increases rapidly for approx. 24 hr post-feeding, rising from -9 ~1 at feeding to -23 ~1 at day 1 (Fig. 3). The volume continues to increase until a maximum ( - 33 ~1) is reached on day 3. From day 3 to day 10 the expressible haemolymph volume remains at this relatively high level, at
volume shows systematic changes during larval-adult development but and temporally different from the ecdysteroid titre (compare Fig. 2).
522
C.G. H. STEEL er al.
which time it begins to decrease gradually and steadily until ecdysis on day 21. The volume at day 20 is _ 15 /.ll. If haemolymph volume changes were responsible for changes in the ecdysteroid titre, then an increase in the haemolymph volume should not result in an increase in the ecdysteroid titre but rather a decrease. The first increase in the ecdysteroid titre (immediately after the blood meal) occurs during a time of increasing expressible haemolymph volume (Figs 1 and 3). Therefore, the rate of appearance of ecdysteroids in the haemolymph at this time is actually higher (rather than lower) than the titre data alone might suggest. Similarly, the large increase in ecdysteroid titre between days 7 and 13 (greater than 6 fold) occurs 2 a time when the expressible haemolymph volume diminishes by only -25% (Figs 2 and 3). These data indicate that ecdysteroid synthesis and a real increase in the ecdysteroid titre has occurred at these times. Therefore, with respect to the changing volumes of haemolymph in Rhodnius during the last larval instar, the two increases in the haemolymph ecdysteroid titre are not due to a dilution or ~on~ntration of existing ecdysteroids but, most likely, to changes in the rates of synthesis and/or excretion/catabolism of these steroid hormones. Ecdysteroid excretion
The possibility of excretory processes affecting or modulating the ecdysteroid titre was probed by determining the ecdysteroids present in either urine excreted during the early part of the larval stadium (O-4 hr) or faeces excreted by late-in&u (days IO and 16) larvae. At the time of the initial increase in the ecdysteroid titre between O-4 hr after the blood meal, intense post-prandial diuresis is occurring: w 100-130 ~1 of urine is excreted per animal, which is equal to about 3O#% of the total blood volume consumed. An analysis of the ecd3teroid RIA activity in methanolic extracts of this urine revealed background levels of RIA activity, indicating that there was no apparent excretion of ecdysteroids. In contrast to the apparent absence of ecdysteroid excretion during the period of the first increase in the ecdysteroid titre, faeces expressed from larvae aged 10 and 16 days (when the ecdysteroid titre is dramatically increasing and decreasing, respectively) exhibit substantial levels ( 2 150 ng/rectum) of ecdysteroid RIA activity. When the ecdysteroid RIA activity in the faeces of Iarvae aged 10 or 16 days is compared to the ecdysteroid RIA activity in the haemolymph (assuming expressible haemolymph volume is ~45% of total volume), about 75% of the total body ecdysteroid RIA activity is in the faeces. These data indicate that (1) excretion of ecdysteroids is occurring at a dramatic rate from the head~ritical period onwards, but is low between feeding and the critical period and (2) that the rate of synthesis of ecdysteroids during the second increase in ecdysteroid titre is occurring at an even more dramatic rate than indicated by the haemolymph titre data alone. lde~tl~eation of ecdysteroids
Qualitative and quantitative analyses of the haemolymph ecdysteroid RIA activity from day 13 animals
revealed the presence of only ecdysone and 20hydroxy-ecdysone. Ratio analysis by TLC showed that 20-hydroxy-ecdysone was the predominant haemolymph ecdysteroid. Confirmation of the TLC analysis was afforded by normal and reverse-phase analytical HPLC (SMITHet al., 1979). The ratio of ecdysone:20hydroxy-ecdysone determined by normal (Zorbax Sil) and reverse (FBondapak Cls) phase separation was 1: 3.7 and 1: 3.4, respectively. Chemical identification of the putative ecdysteroids was by mass spectrometry (SMITHer al., 1979) and revealed that the endogeneous haemolymph ecdysteroids were ecdysone and 20hydroxy-ecdysone. No attempt was made to identify the ecdysteroid RIA active material in the faeces. However, given the low affinity of most naturally occurring ecdysteroid metabolites and conjugates for the D-10 antiserum used in these investigations, it is probable that the large amounts of RIA active material in the faeces are predominantly ecdysone and 20-hydroxy-ecdysone. DISUNION In recent years ecdysteroid titres have been determined during the development of various species of insects (references in DEAN et al., 1980). However, most of the insects investigated were holometabolous and even for these (except for cathodes and ~anducu) substantial information is lacking regarding the timing and physiological basis of tissue responses to the moulting hormone. Even less is understood about the mechanisms by which the ecdysteroid titre is regulated. By contrast, information does exist for Rhodnius {WI~LESWOR~, 1964; &EEL, 1978, 1981) and we take this opportunity to correlate the haemolymph ecdysteroid titre with these data on the physiology of cellular growth and development and explore the mechanisms for controlling the ecdysteroid titre, i.e. neurosecretion and excretion. During Rhadnias larva-adult development, there are two significant increases in the ecdysteroid titre, a small one occurring immediately following feeding, reaching a maximum of -70 ng/ml and a second dramatic increase later in the instar reaching SO-100 times that amount. The principal ecdysteroids are 2~hydroxy-ecdysone and ecdysone, with 20-hydroxyecdysone being the predominant molecule. This is consistent with the ascribed function of 20-hydroxyecdysone as the moulting hormone. These fluctuations in the titre are, in part, a result of ecdysone synthesis by the prothoracic glands since changes in haemolymph volume that occur during development do not affect the ecdysteroid titre significantly. As has been concluded from ecdysteroid titres in other insects (GILBERTet al., 1980), the two increases in the titre during the last-larval stadium probably have specific functions, both of which are required for development and metamorphosis to the adult. Relationship of the titre to the physiology of larval tissues
The finding of a negligible level of ecdysteroids in the haemolymph of unfed Rhodnius is consistent witb the classical interpretation that this state is one of developmental arrest resembling diapause (WIGGLESWORTH, 1964). The taking of a blood meal by the
Ecdysteroid titres in Rhodnius last-stage larva initiates adult development and results in characteristic cytological changes in the epidermis. fat body and abdominal muscle within v 6 hr (WI~LESWO~TH, 1957). Nucfeolar enlargement, mitochondrial swelling and increased cytoplasmic RNA, collectively termed ‘activation’, are the first overt changes that occur and have been interpreted as indicating a restored capacity for protein synthesis in these tissues (%GGLESwORTH, 1957). The injection of ecdysone into unfed Rhodnius has been shown to ‘activate’ epidermis and muscle in a manner similar to feeding. Since the ecdysteroid titre increases rapidly to a low, but signi~~nt, level upon feeding, it appears that the activation of these tissues is actually elicited by 20-hydroxy-ecdysone. When a larva is decapitated 24 hr after feeding, the cells regress to an inactive state (WIGGL~WOR~, 1934), suggesting that maintenance of the activated state of the tissue requires a continuous exposure to ecdysteroids. At day 7-8 of the last instar of animals reared at 24°C mitosis is evident in the abdominal epidermis, while at 28°C this occurs at about day S-6. Mitosis is not thought to be a direct response to ecdysteroids, but occurs only in cells which have previously undergone ‘activation’ ~~~L~WOR~ 1940, 1963).Mitotic activity therefore occurs at the end of the sustained plateau phase of the ecdysteroid titre. The time during the last instar when the epidermal cells undergo a change in commitment, i.e. are reprogrammed to secrete adult rather than larva1 cuticle, is usually close to the time of mitosis: in i2/lat&ca (RIDDIFORD, 1976, 1978; WIELGUS et al., 1979) and Calpodes (DEANet al., 1980), this change in commitment occurs just prior to mitosis, whereas in Galleria it occurs after mitosis (HWANG-HSU et ai., 1979). In Rhodnius, injections of large doses of 20-hydroxyecdysone into larvae from day 1 to day 5 of the lastlarval instar results in the deposition of larval cuticie, while injections after day 5 result in adult cuticle deposition (C. G. H. STEEL, unpublished). Similarly, the cuticular response to application of farnesyl methyl ether drops precipitously at days 4-5 (K. G. DAVEY,personal communication). It appears, therefore, that there is a change in commitment in Rhodnius as in these holometabolous insects, and this commitment occurs at about day 4-5 of the last instar, a time just preceding mitosis. In ~anducu (BOLLENBACHERet al., 1975; RIDDIFORD,1976, 1978) and Caipodes (DEAN et al., 1980) commitment is evoked by a subtfe peak in the haemolymph ecdysteroid titre about midway through the instar. However, an eedy steroid peak was not found in Rhod~ias at this time. It is possible that the change in commitment in Rhodnius is elicited not by a discrete ecdysteroid peak but by some consequence of proionged exposure of the tissues to the Iow levels of ecdysteroids seen between feeding and day 5. Nevertheless, the change in commitment is just as abrupt as it is in those species in which it is associated with a discrete peak in the ecdysteroid titre. During the large peak in the ecdysteroid titre from days 7-18 the prothoracic gland cells undergo cytological changes indicative of substantial physiological activity (W~~L~WOR~, 1952). At the time of this increase, apolysis occurs in the abdominal tergites (WIGGLESWORTH,1940), while other regions of the
523
animal undergo apolysis at slightly different times (WIGGLESWORTH, 1973). During this time, the oenocytes swell in a manner suggestive of secretory activity ~I~L~WOR~, 1933). These cells have been implicated in the production of lipoproteins for the new cuticle (WIGGLESWORTH,1970) but whether this occurs in response to the increase in the ecdysteroid titre is not known. At 28°C epicuticle formation occurs on day 13 and exocuticle secretion commences on day 14-15, with epicuticle becoming darkened on day 18 (WIGGLESWORTH,1933). Thus, epicuticle secretion occurs at the peak ecdysteroid titre and the subsequent cuticular changes occur during a period of declining ecdysteroid titre, suggesting that these processes were ehcited by moulting hormone. Role of the brain In R~odnius, the prothoracic glands are stimulated by the release of PTTH which is presumably synthesized by medial neurosecretory cells of the brain ~I~L~WOR~, 1940, 1952). The occurrence of a head-critical period at days 6-8 after feeding at 24°C (WIGGLESWORTH, 1934), (or 5-6 days at 28°C; STEEL, 1982), has been interpreted as indicating a progressive activation of the prothoracic glands by PTTH (WI~L~WOR~, 1957, 1964). However, this concept of a single head critical period in Rhodnius does not agree with the two increases in the haemolymph ecdysteroid titre observed in this insect. Rather these results indicate the stimulation of the prothoracic glands by PTTH on two separate occasions, and by implication, two periods of PITH release. That PTTH release is necessary within 24hr of feeding to elicit the initial ecdysteroid-dependent tissue changes is supported by a number of studies, The ‘activation’ of tissues by ecdysteroids is abolished by decapitation at 24 hr after feeding (WKXLESWORTH, 1934), and when brain fragments containing medial neurosecretory cells are implanted into decapitated larvae, the ‘activation’ of the tissues is restored (WIGGLESWORTH,1940). Light and electron microscope studies of the Rhod~ius brain (STEEL and HARM.SEN, 1971; MORRIS and STEEL. 1975, 1977) have revealed a variety of structural changes in the medial neurosecretory cells within minutes of feeding which are indicative of neurohormone release. Recently, electrical recordings from the axons of these cells of newly fed last-instar larvae have revealed the rapid appearance of a bursting firing pattern indicative of hormone release (ORCHARDand STEEL, 1980). This pattern disappears at 2 hr after a blood meal, with only a low frequency continuous firing pattern persisting after this time. This change in electrical activity would suggest a drastic attenuation of hormone release at about 2 hr which is the time that the increasing haemolymph ecdysteroid titre reaches its plateau. Therefore, these data support the concept of an initial period of PTTH release occurring from 0 to 2 hr after feeding. This would result in stimulation of ecdysteroid production, leading to the reported increase in the ecdysteroid titre and the tissue changes discernible within 6 hr. The bursting firing pattern that is associated with the release of PTTH upon feeding reappears at a time just before the head critical period (ORCHARDand STEEL,1980). Here bursting activity persists for 24 hr
C. G. H. STEELer al.
524
and the ecdysteroid titre begins to increase during this time (STEEL,1982). The coiticidence of this bursting activity and the head critical period was shown by decapitating animals every 4 hr during this period (STEEL,1982); animals decapitated before this period failed to moult. This second period of bursting activity by the medial neurosecretory cells and the subsequent increase in the ecdysteroid titre supports our postulate that this is the second period of PTTH release during larval-adult development in Rhodnius. The above considerations indicate that PTTH release from the brain occurs on two distinct occasions, the first marking the commencement of the plateau phase (at O-2 hr), the second marking the commencement of the large peak (at 5-6 days). However, additional control mechanisms must also exist, for the changes in haemolymph titre following the two releases differ both temporally and quantitatively. The only such mechanism examined in the present paper is the role of excretion. The finding that ecdysteroid excretion in Rhodnius occurs at high haemolymph titres but not at low is the converse of findings with Locusta (HOFFMAN et al., 1974; FEYEREISEN et al., 1976). Since the amounts of ecdysteroid excreted when the titre is increasing (day 10) and decreasing (day 16) are similar, the role of excretion is clearly more subtle than merely the elimination of excess ecdysteroid prior to ecdysis. The observed pattern of ecdysteroid excretion is consistent with the model developed by MADDRELL and GARDINER (1980) to explain the pattern of excretion of other small organic solutes. At times other than diuresis, this model requires selective resorption of such solutes and thereby raises the novel possibility that stage-dependent ecdysteroid excretion may result from control of ecdysteroid resorption from the Malpighian tubule lumen. Acknowledgements-This research was supported by an operating grant from the Natural Sciences and Engineering Council of Canada (A 6669) to C. G. H. Steel, NIH grant (AM-30118) to L. I. Gilbert and NIH (NS-15387) and USDA (59-2167) grants to W. E. Bollenbacher. The expert technical assistance of K. Prasad is gratefully acknowledged.
REFERENCES BOLLENBACHER W. E., VEDECK~S W. V., GILBERTL. I. and O’CONNOR J. D. (1975) Ecdysone titres and prothoracic gland activity during the larval-pupal development of Manduca sexta. Deal. Biol. 44, 4653.
BORSTD. W. and O’CONNORJ. D. (1974) Trace analysis of ecdysones by gas-liquid chromatography, radioimmunoassay and bioassay. Steroids 24, 637-656. BUXTONP. A. (1930) The biology of a blood-sucking bug, Rhodnius prolixus.’ Trans R. eni. Sot. Lond. 78, 227-236.
DEANR. L.. BOLLENBACHER W. E., IAXKE M., SMITHS. L. and GILBERTL. I. (1980) Haemolymph ecdysteroid levels and cellular events in the intermoult/moult sequence of Calpodes ethlius. J. Insect Physiol. 26, 267-280.
FEYEREISEN R., LAGEUXM. and HOFFMANJ. A. (1976) Dynamics of ecdysone metabolism after ingestion and injection in Locusta migratoria. Gen. camp. Endocr. 29, 3 19-327.
GILBERTL. I., GOODMANW. and BOLLENBACHER W. E. (1977) Biochemistry of regulatory lipids and sterols in insects. Int. Rev. Biochem. 14, l-50. GILBERTL. I., BOLLENBACHER W. E., GOODMAN W., SMITH S. L., AGUI N., GRANGER N. and SEDLAKB. J. (1980)
Hormones controlling insect metamorphosis.
Rec. Prog.
Hormone Res. 36, 401449.
GRINGORTEN J. L. and FRIENDW. G. (1979) Haemolymphvolume changes in Rhodnius prolixus during flight. J. exp. Biol. 83, 325-333.
HOFFMANJ. A., KOOLMANJ., KARL~N P. and JOLY P. (1974) Molting hormone titer and metabolic fate of injected ecdysone during the fifth larval instar and in adults of Locusta migratoria (Orthoptera). Gen. camp. Endocr. 22,90-97.
HWANG-HSU K., REDDY G., KUMARANA. K., BOLLENBACHERW. E. and GILBERTL. I. (1979) Correlations between juvenile hormone esterase activity, ecdysone titre and cellular reprogramming in Galleria mellonella. J. Insect Physiol. 25, 105-111. KRUSKALW. H. and WALLISW. A. (1952) The use of ranks in one-criterion variance analysis. J. Am. starist. Ass. 47, 583-621.
LOCKEM. and SMITHD. S. (Eds.) (1980) Insecr Biology in the Future. Academic Press, New York. MADDRELLS. H. P. (1964) Excretion in the blood-sucking bug, Rhodnius prolixus Stil II. The normal course of diuresis and the effect of temperature. J. exp. Biol. 41, 163-176. MADDRELLS. H. P. and GARDINERB. 0. C. (1980) The retention of amino acids in the haemolymph during diuresis in Rhodnius. J. exp. Biol. 87, 315-329. MORRISG. P. and STEELC. G. H. (1975) Ultrastructure of neurosecretory cells in the pars intercerebralis of Rhodnius prolixus (Hemiptera). Tissue Cell 7, 73-90. MORRISG. P. and STEELC. G. H. (1977) Sequence of ultrastructural changes induced by activation in the posterior neurosecretory cells in the brain of Rhodnius prolixus with special reference to the role of lysosomes. Tissue Cell 9, 547-561. ORCHARDI. and STEELC. G. H. (1980) Electrical activity of neurosecretory axons from the brain of Rhodnius prolixus: relation of changes in the pattern of activity to endocrine events during the moulting cycle. Brain Res. 191, 53-65. RIDDIFORDL. M. (1976) Hormonal control of insect epiderma1 cell commitment in uitro. Nature, Lond. 259, 115-1.17. RIDD~FORD L. M. (1978) Ecdysone-induced change in cellular commitment of the epidermis of the tobacco hornworm, Manduca sexta, at the initiation of metamorphosis. Gen. camp. Endocr. 34,438-446. S&H S. L., BOLLENBACHER W. E., CCI~PERD., SCHLEYER H.. WIELGUSJ. J. and GILBERTL. I. (1979) Ecdvsone 2&monooxygenase: characterization of an insect -cytochrome P-450 dependent steroid hydroxylase. Molec. Cell. Endocrin. 15, 111-133. SAL C. G. H. (1978) Nervous and hormonal regulation of, neurosecretory cells in the insect brain. In Comparatiue Endocrinology (Edited by GAILLARDP. J. and BOER H. H. ), pp. 327-330. Elsevier, Amsterdam. STEELC. G. H. (1982) Parameters and timing of synthesis, transport and release of neurosecretion ;n thk insect brain. In Neurosecretion (Edited bv FARNERD. S. and LEDERISK.), pp. 223-233. Plenum Press, New York. STEELC. G. H. and HARMSENR. (1971) Dynamics of the neurosecretory system in the brain of an insect, Rhodnius prolixus, during growth and molting. Gen. camp. Endocr. 17, 125-141.
URIBEC. (1927) On the biology and life history of Rhodnius prolixus Stahl. J. Parasit. 13, 129-136. WIELGUSJ., BOLLENBACHER W. E. and GILBERTL. 1. (1979) Correlations between epidermal DNA synthesis and haemolymph ecdysteroid titre during the last larval instar of the tobacco homworm, Manduca sexta. J. Insect Physiol. 25, 9-16.
WIGGLESWORTH V. B. (1933) The physiology of the cuticle and of ecdysis in Rhodnius prolixus (Triatomidae, Hemi-
Ecdysteroid titre ‘s in Rhodnius ptera); with special reference to the function of the oenocytes and of the dermal glands. Q. J. microsc. Sci. 76, 296318.
WIGGLESWORTH V. B. (1934) The physiology of ecdysis in Rhodnius prolixus (Hemiptera). II. Factors controlling moulting and ‘metamorphosis’. Q. J. microsc. Sci. 77, 191-222. WIGGLESWORTH V. B. (1940) The determination of characters at metamorphosis in Rhodnius prolixus (Hemiptera). J. exp. Biol. 17, 201-222. WIGGLESWORTH V. B. (1952) The thoracic gland in Rhodnius prolixus (Hemiptera) and its role in moulting. J. exp. Biol. 29, 561-570.
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WIGGLESWORTH V. B. (1957) The action of growth hormones in insects. Symp. Sot. exp. Biol. 11, 204227. WIGGLESWORTH V. B. (1963) The action of moulting hormone and juvenile hormone at the cellular level in Rhodnius prolixus. J. exp. Biol. 40, 231-245. WIGGLESWORTII V. B. (1964) The hormonal regulation of growth and reproduction in insects. Adc. Insect Physiol. 2, 247-336. WIGGLESWORTH V. B. (1970) Structural lipids in the insect cuticle and the function of the oenocytes. Tissue Cell 2, 155-179. WIGGLESWORTH V. B. (1973) The significance of “apolysis” in the moulting of insects. J. Ent. (A). 47, 141-149.
~22-1910/82/o60s19-07~3~~/0 0 1982 Pergamon Press Od
HAEMOLYMPH ECDYSTEROID TITRES LARVAL-ADULT DEVELOPMENT
DURING
IN
R~O~~~US ~RO~~X~S~ CORRELATIONS WITH MOULTING HORMONE ACTION AND BRAIN NEUROSECRETORY CELL ACTIVITY C. 6. H. STEEL*, W. E. BOLLENBACHER~, S. L. SMITH$and L. I. GI~BERH *Department of Biology, York University, Downsview, Ontario M3J lP3, Canada; fDepartment of Zoology, University of North Carolina, Chapel Hill, NC 27.514, U.S.A, and ~~part~nt of Biological Sciences, Bo~lj~g Green State University, Bowling Green. OH 43403. U.S.A. (Received 23 October 1981) Abstract-A haemolymph ecdysteroid titre of the fifth (last)-larval instar of the hemipteran, Rhodnius prolixus has been determined by radioimmunoassay. During the last-larval stadium the ecdysteroid titre increases from a negligible level in the unfed insect to a detectable level within minutes following a blood meal. The titre reaches a plateau of + 50-70 ng/ml at 3-4 hr and this level is maintained until day 5-6, the time of the head-critical period in Rkodnjus. At the head-critical period the titre begins to increase again, this time dramatically, reaching a peak of -3500 ng/ml at day 13. From day 14 to ecdysis (day 21) the titre declines to a low level, _ 30 r&ml. Basal levels of ecdysteroids, + 15 ng/mI, were detectable in young adult males and females. A survey of haemoiymph volumes during the last-larval instar indicates that the changes in the ecdysteroid titre reflect changes in the rates of ecdysteroid synthesis, and not changes in haemolymph volume. Excretion of ecdysteroids varies systematically during the instar, suggesting that control of ecdysteroid excretion may be important in regulation of the haemolymph titre. Qualitative analysis of the haemolymph ecdysteroid,RIA activity revealed the presence of only ecdysone and 20-hydrox~e~dysone. For the large peak preceding larval-adult ecdysis, 2~hydroxy-ecdysone was the predominant hormone. These rest&s indicate that there may be two periods of release of prothoracicotropic hormone (PTTH) from the brain in Rk~n~us, one immediately following the biood meal and the second on day 5 or 6. The significance of these times of PTTH release is discussed in relation to classical evidence of the timing of mouiting hormone action, the response of target tissues, and with more recent findings on the timing of release of neurosecretory material from the brain of Rkodnius during moulting. Key Word Index: Ecdysteroids, moulting, neurosecretion. prothoracicotropic hormone, Rkodnius
with the pioneering studies of Wigglesworth, research on the blood-sucking bug, Rho&us prolixus, has contributed substantially to our understanding of the endocrine regulation of growth and development in insects. Early observations that one cycle of moulting was initiated by a blood meal revealed that Rhodnius was an ideal insect for investigating the physiology of moulting on a precise timetable (URIBE, 1927; BUXTON,1930). Its usefulness as an experimental animal was enhanced by the fact that this bug could be surgically manipulated (e.g. decapitation, parabiosis) without the dangers of interference with continued feeding. The numerous publications on Rho~nius by Wi~lesworth (see LOCKEand SMITH 1980) provided considerable insight into the diverse tissue changes occurring during insect growth and development, and the endocrine regulation of these changes by hormones from the brain, corpora allata and prothoracic glands. It is ironic, considering the role of Rhodnius in establishing the dogma of the endo~rinolo~~l control of insect moulting and metamorphosis, that the titres of the hormones involved have not yet been determined. ~O~NC~G
The present paper reports an analysis of the ecdysteroid titre for the fifth (last)-larval instar of Rhodnius. This study details both quantitative and qualitative aspects of the titre. The significance of the titre is discussed in relation to classical studies on the secretion and effects of the moulting hormone on various tissues (see WIGGLESWORTH,1957, 1964), together with more recent studies on the role of neuros~retion from the brain in the control of moulting in Rhodnius (STEEL,1978. 1982). MATERIALS
AND MXTI-IODS
Animals ~~~~iu~ proiixus, derived from the culture used by Wigglesworth, were raised at 28 t_ 1°C an$ about 70% r.h. Fifth-instar larvae were timed from the blood meal (day 0). At this temperature adult ecdysis occurs 5 21 days after the blood meal. Wigglesworth’s early work employed Rhodn~us reared at 24°C with the fifth-larval stadium lasting -26 days (WIGGLESWORTH, 1934). To facilitate comparisons betweeni these investigations and the earlier studies of Wiggles:, worth, the timing given at 24°C has been adjusted, t&
519
520
C. G. H.
28°C in proportion to the interval from feeding to ecdysis at the two temperatures. With this procedure the assumption is made that all developmental events in the fifth-instar larvae are equally temperature sensitive. Although the adjusted times are regarded as approximate, this method is accurate for all events whose timing is known at both temperatures and indicates that the overall moulting cycle has a Qlo close to 2. Co~leetion of huemo~ymph, urine u~~eces
Haemolymph was expressed from the animals through a tibia1 cut and collected with a calibrated glass capillary. With moderate finger pressure on the animal, haemolymph was collected until it ceased to emerge in a steady stream. The volume of haemolymph collected in this manner is referred to as the expressible haemolymph volume and is used as a relative index of the haemolymph volume in an animal. Actual haemolymph volumes have been determined only for the early portion of the last-larval stadium (MADDRELL and GARDINER, 1980) in which the expressible haemolymph volume is a fairly constant 45% of the total. A similar proportion is expressible in the adult (GRINGORTENand FRIEND, 1979). This volume is therefore useful as an index of trends, although not of absolute volumes. During the profuse diuresis which occurs during the first few hours following feeding, fifth-instar Rhodnius excrete a watery urine equivalent to about three times their unfed weight (MADDRELL,1964). The total volume of urine produced by individual animals during diuresis was collected on filter paper and the volume of the urine determined from the loss in v$&ht of the animal. The filter paper was permitted to dry, extracted with methanol, and the methanolic extract assayed for ecdysteroids. Following diuresis, evacuation of the rectum in Rhodnius was carried out by handling the animals. Rectal contents were collected for extraction and assay of ecdysteroids.
STEELet aI.
previously (SMITHet al., 1979). Final identification of the ecdysteroids was by mass spectrometry.
RESULTS Haemolymph ecdysteroid titre
The taking of a blood meal by the larvae of Rhodof arrested development and the initiation of a moulting cycle. At feeding, the fifth&star larva ingests blood equal to * 10 times its weight in - 10 min. The amount of ecdysteraid RIA activity in the haemolymph of an unfed animal is negligible, but within minutes after the larva begins to feed the ecdysteroid titre begins to increase. The titre increases linearly and rapidly for 3-4 hr, at which time the titre is 5 70 ng/ml haemolymph (Fig. 1). This significant (P < 0.01) increase in the ecdysteroid titre between 0 and 3 hr may indicate the time of the first release of PTTH for larval-adult development in Rhodnius (see Discussion). From 4 to 24 hr post-feeding the titre decreases gradually to a level of -50 ng/ml haemolymph. This apparent decrease between 4 and 24 hr is not statistically significant (P > 0.5). From day 1 of the last instar to day 5 the ecdysteroid titre remains constant at w 50 ng/ml, but between days 5 and 7 (during the time of the classical head critical period} the titre begins to increase again, reaching its highest level (about 3~n~rnl~ during the 21-day stadium on day 13 (Fig. 2). This latter increase in the titre is probably in response to the release of PTTH at this head-critical period (It should be noted that further analyses at day 13-14 have resulted in mean values of 78OOng/ml). The titre remains relatively unchanged during days 13 and 14 and then drops dramatically by day 16 (- 800ng/ml) reaching a low level of 30ng/ml on day 20. Up to 3 days post-ecdysis, adult males and females exhibit a nius results in the termination
Ecdysteroid titre and chemistry
Ecdysteroids were extracted from haemolymph, urine and faeces by homogenization in methanol. After centrifugation at 8000 g for 10 min, the ecdysteroids in the methanolic extracts were quantified by radioimmunoassay (RIA) with D-10 antiserum which binds ecdysone and 20-hydroxy-ecdysone equally (BORSTand O’CONNOR,1974; GILBERT et al., 1977). RIA activity is expressed in 20-hydroxy-ecdysone equivalents since this ecdysteroid was used as the standard. The quantitative accuracy of the RIA was ascertained by the addition of 20-hydroxy-ecdysone as an internal standard as well as by purification of the ecdysteroids. All samples were assayed in duplicate, usually at 3-5 different concentrations. The labelled ligand for the RIA was [23,24-3H]-ecdysone ( - 57 Ci/mmol). Statistical analyses of the titre data were by Student’s t-test and non-parametric analysis of variance (KRUSKALand WALLI~,1952). Chemical analysis of the haemol~ph ecdysteroids was carried out by thin layer chromato~aphy (TLC), analytical normal and reverse phase high pressure liquid chromatography (HPLC) and RIA as described
Fig. 1. H~molymph ecdysteroid titres shortly after the initiation of larval-adult development at feeding. Ecdysteroids are undete~tabie prior to feeding but have increased to significant levels within 30 min. The level reached at 3-4 br is maintained for 5 days.
tines in Rhodnius
Ecdysteroid
0 J-0
J-4
_.-.-.
521
\
w* /6
B
I’0
l-2
1’4
Days after feeding
lb
‘-._
l’s
.-.
lo
v3
+ ecdysis
Fig. 2. Haemolymph ecdysteroid titres during larval-adult development. Standard errors for some of the samples are smaller than the point symbols. Time axis is interrupted at ecdysis and the samples taken thereafter are grouped by time since ecdysis.
marginally (Fig. 2). Haemolymph
detectable
ecdysteroid
titre of _ 15 ng/ml
volume
Due to the nature of the induction of the moult cycle in Rhodnius, the distinct possibility exists that the haemoiymph volume in a larva may vary dramatically following the blood meal, and this may give rise to an apparent increase or decrease in the ecdysteroid titre depending on whether haemolymph volume increases or decreases. To examine the possibility that fluctuations in the haemolymph volume may result in
Fig. 3. Expressible haemolymph these are both qualitatively
an apparent change in the haemolymph ecdysteroid titre, the expressible haemolymph volumes during the last-larval stadium have been determined and these volumes compared to the ecdysteroid titre during this period. During the last-larval stadium of Rhodnius, the expressible haemolymph volume increases rapidly for approx. 24 hr post-feeding, rising from -9 ~1 at feeding to -23 ~1 at day 1 (Fig. 3). The volume continues to increase until a maximum ( - 33 ~1) is reached on day 3. From day 3 to day 10 the expressible haemolymph volume remains at this relatively high level, at
volume shows systematic changes during larval-adult development but and temporally different from the ecdysteroid titre (compare Fig. 2).
522
C.G. H. STEEL er al.
which time it begins to decrease gradually and steadily until ecdysis on day 21. The volume at day 20 is _ 15 /.ll. If haemolymph volume changes were responsible for changes in the ecdysteroid titre, then an increase in the haemolymph volume should not result in an increase in the ecdysteroid titre but rather a decrease. The first increase in the ecdysteroid titre (immediately after the blood meal) occurs during a time of increasing expressible haemolymph volume (Figs 1 and 3). Therefore, the rate of appearance of ecdysteroids in the haemolymph at this time is actually higher (rather than lower) than the titre data alone might suggest. Similarly, the large increase in ecdysteroid titre between days 7 and 13 (greater than 6 fold) occurs 2 a time when the expressible haemolymph volume diminishes by only -25% (Figs 2 and 3). These data indicate that ecdysteroid synthesis and a real increase in the ecdysteroid titre has occurred at these times. Therefore, with respect to the changing volumes of haemolymph in Rhodnius during the last larval instar, the two increases in the haemolymph ecdysteroid titre are not due to a dilution or ~on~ntration of existing ecdysteroids but, most likely, to changes in the rates of synthesis and/or excretion/catabolism of these steroid hormones. Ecdysteroid excretion
The possibility of excretory processes affecting or modulating the ecdysteroid titre was probed by determining the ecdysteroids present in either urine excreted during the early part of the larval stadium (O-4 hr) or faeces excreted by late-in&u (days IO and 16) larvae. At the time of the initial increase in the ecdysteroid titre between O-4 hr after the blood meal, intense post-prandial diuresis is occurring: w 100-130 ~1 of urine is excreted per animal, which is equal to about 3O#% of the total blood volume consumed. An analysis of the ecd3teroid RIA activity in methanolic extracts of this urine revealed background levels of RIA activity, indicating that there was no apparent excretion of ecdysteroids. In contrast to the apparent absence of ecdysteroid excretion during the period of the first increase in the ecdysteroid titre, faeces expressed from larvae aged 10 and 16 days (when the ecdysteroid titre is dramatically increasing and decreasing, respectively) exhibit substantial levels ( 2 150 ng/rectum) of ecdysteroid RIA activity. When the ecdysteroid RIA activity in the faeces of Iarvae aged 10 or 16 days is compared to the ecdysteroid RIA activity in the haemolymph (assuming expressible haemolymph volume is ~45% of total volume), about 75% of the total body ecdysteroid RIA activity is in the faeces. These data indicate that (1) excretion of ecdysteroids is occurring at a dramatic rate from the head~ritical period onwards, but is low between feeding and the critical period and (2) that the rate of synthesis of ecdysteroids during the second increase in ecdysteroid titre is occurring at an even more dramatic rate than indicated by the haemolymph titre data alone. lde~tl~eation of ecdysteroids
Qualitative and quantitative analyses of the haemolymph ecdysteroid RIA activity from day 13 animals
revealed the presence of only ecdysone and 20hydroxy-ecdysone. Ratio analysis by TLC showed that 20-hydroxy-ecdysone was the predominant haemolymph ecdysteroid. Confirmation of the TLC analysis was afforded by normal and reverse-phase analytical HPLC (SMITHet al., 1979). The ratio of ecdysone:20hydroxy-ecdysone determined by normal (Zorbax Sil) and reverse (FBondapak Cls) phase separation was 1: 3.7 and 1: 3.4, respectively. Chemical identification of the putative ecdysteroids was by mass spectrometry (SMITHer al., 1979) and revealed that the endogeneous haemolymph ecdysteroids were ecdysone and 20hydroxy-ecdysone. No attempt was made to identify the ecdysteroid RIA active material in the faeces. However, given the low affinity of most naturally occurring ecdysteroid metabolites and conjugates for the D-10 antiserum used in these investigations, it is probable that the large amounts of RIA active material in the faeces are predominantly ecdysone and 20-hydroxy-ecdysone. DISUNION In recent years ecdysteroid titres have been determined during the development of various species of insects (references in DEAN et al., 1980). However, most of the insects investigated were holometabolous and even for these (except for cathodes and ~anducu) substantial information is lacking regarding the timing and physiological basis of tissue responses to the moulting hormone. Even less is understood about the mechanisms by which the ecdysteroid titre is regulated. By contrast, information does exist for Rhodnius {WI~LESWOR~, 1964; &EEL, 1978, 1981) and we take this opportunity to correlate the haemolymph ecdysteroid titre with these data on the physiology of cellular growth and development and explore the mechanisms for controlling the ecdysteroid titre, i.e. neurosecretion and excretion. During Rhadnias larva-adult development, there are two significant increases in the ecdysteroid titre, a small one occurring immediately following feeding, reaching a maximum of -70 ng/ml and a second dramatic increase later in the instar reaching SO-100 times that amount. The principal ecdysteroids are 2~hydroxy-ecdysone and ecdysone, with 20-hydroxyecdysone being the predominant molecule. This is consistent with the ascribed function of 20-hydroxyecdysone as the moulting hormone. These fluctuations in the titre are, in part, a result of ecdysone synthesis by the prothoracic glands since changes in haemolymph volume that occur during development do not affect the ecdysteroid titre significantly. As has been concluded from ecdysteroid titres in other insects (GILBERTet al., 1980), the two increases in the titre during the last-larval stadium probably have specific functions, both of which are required for development and metamorphosis to the adult. Relationship of the titre to the physiology of larval tissues
The finding of a negligible level of ecdysteroids in the haemolymph of unfed Rhodnius is consistent witb the classical interpretation that this state is one of developmental arrest resembling diapause (WIGGLESWORTH, 1964). The taking of a blood meal by the
Ecdysteroid titres in Rhodnius last-stage larva initiates adult development and results in characteristic cytological changes in the epidermis. fat body and abdominal muscle within v 6 hr (WI~LESWO~TH, 1957). Nucfeolar enlargement, mitochondrial swelling and increased cytoplasmic RNA, collectively termed ‘activation’, are the first overt changes that occur and have been interpreted as indicating a restored capacity for protein synthesis in these tissues (%GGLESwORTH, 1957). The injection of ecdysone into unfed Rhodnius has been shown to ‘activate’ epidermis and muscle in a manner similar to feeding. Since the ecdysteroid titre increases rapidly to a low, but signi~~nt, level upon feeding, it appears that the activation of these tissues is actually elicited by 20-hydroxy-ecdysone. When a larva is decapitated 24 hr after feeding, the cells regress to an inactive state (WIGGL~WOR~, 1934), suggesting that maintenance of the activated state of the tissue requires a continuous exposure to ecdysteroids. At day 7-8 of the last instar of animals reared at 24°C mitosis is evident in the abdominal epidermis, while at 28°C this occurs at about day S-6. Mitosis is not thought to be a direct response to ecdysteroids, but occurs only in cells which have previously undergone ‘activation’ ~~~L~WOR~ 1940, 1963).Mitotic activity therefore occurs at the end of the sustained plateau phase of the ecdysteroid titre. The time during the last instar when the epidermal cells undergo a change in commitment, i.e. are reprogrammed to secrete adult rather than larva1 cuticle, is usually close to the time of mitosis: in i2/lat&ca (RIDDIFORD, 1976, 1978; WIELGUS et al., 1979) and Calpodes (DEANet al., 1980), this change in commitment occurs just prior to mitosis, whereas in Galleria it occurs after mitosis (HWANG-HSU et ai., 1979). In Rhodnius, injections of large doses of 20-hydroxyecdysone into larvae from day 1 to day 5 of the lastlarval instar results in the deposition of larval cuticie, while injections after day 5 result in adult cuticle deposition (C. G. H. STEEL, unpublished). Similarly, the cuticular response to application of farnesyl methyl ether drops precipitously at days 4-5 (K. G. DAVEY,personal communication). It appears, therefore, that there is a change in commitment in Rhodnius as in these holometabolous insects, and this commitment occurs at about day 4-5 of the last instar, a time just preceding mitosis. In ~anducu (BOLLENBACHERet al., 1975; RIDDIFORD,1976, 1978) and Caipodes (DEAN et al., 1980) commitment is evoked by a subtfe peak in the haemolymph ecdysteroid titre about midway through the instar. However, an eedy steroid peak was not found in Rhod~ias at this time. It is possible that the change in commitment in Rhodnius is elicited not by a discrete ecdysteroid peak but by some consequence of proionged exposure of the tissues to the Iow levels of ecdysteroids seen between feeding and day 5. Nevertheless, the change in commitment is just as abrupt as it is in those species in which it is associated with a discrete peak in the ecdysteroid titre. During the large peak in the ecdysteroid titre from days 7-18 the prothoracic gland cells undergo cytological changes indicative of substantial physiological activity (W~~L~WOR~, 1952). At the time of this increase, apolysis occurs in the abdominal tergites (WIGGLESWORTH,1940), while other regions of the
523
animal undergo apolysis at slightly different times (WIGGLESWORTH, 1973). During this time, the oenocytes swell in a manner suggestive of secretory activity ~I~L~WOR~, 1933). These cells have been implicated in the production of lipoproteins for the new cuticle (WIGGLESWORTH,1970) but whether this occurs in response to the increase in the ecdysteroid titre is not known. At 28°C epicuticle formation occurs on day 13 and exocuticle secretion commences on day 14-15, with epicuticle becoming darkened on day 18 (WIGGLESWORTH,1933). Thus, epicuticle secretion occurs at the peak ecdysteroid titre and the subsequent cuticular changes occur during a period of declining ecdysteroid titre, suggesting that these processes were ehcited by moulting hormone. Role of the brain In R~odnius, the prothoracic glands are stimulated by the release of PTTH which is presumably synthesized by medial neurosecretory cells of the brain ~I~L~WOR~, 1940, 1952). The occurrence of a head-critical period at days 6-8 after feeding at 24°C (WIGGLESWORTH, 1934), (or 5-6 days at 28°C; STEEL, 1982), has been interpreted as indicating a progressive activation of the prothoracic glands by PTTH (WI~L~WOR~, 1957, 1964). However, this concept of a single head critical period in Rhodnius does not agree with the two increases in the haemolymph ecdysteroid titre observed in this insect. Rather these results indicate the stimulation of the prothoracic glands by PTTH on two separate occasions, and by implication, two periods of PITH release. That PTTH release is necessary within 24hr of feeding to elicit the initial ecdysteroid-dependent tissue changes is supported by a number of studies, The ‘activation’ of tissues by ecdysteroids is abolished by decapitation at 24 hr after feeding (WKXLESWORTH, 1934), and when brain fragments containing medial neurosecretory cells are implanted into decapitated larvae, the ‘activation’ of the tissues is restored (WIGGLESWORTH,1940). Light and electron microscope studies of the Rhod~ius brain (STEEL and HARM.SEN, 1971; MORRIS and STEEL. 1975, 1977) have revealed a variety of structural changes in the medial neurosecretory cells within minutes of feeding which are indicative of neurohormone release. Recently, electrical recordings from the axons of these cells of newly fed last-instar larvae have revealed the rapid appearance of a bursting firing pattern indicative of hormone release (ORCHARDand STEEL, 1980). This pattern disappears at 2 hr after a blood meal, with only a low frequency continuous firing pattern persisting after this time. This change in electrical activity would suggest a drastic attenuation of hormone release at about 2 hr which is the time that the increasing haemolymph ecdysteroid titre reaches its plateau. Therefore, these data support the concept of an initial period of PTTH release occurring from 0 to 2 hr after feeding. This would result in stimulation of ecdysteroid production, leading to the reported increase in the ecdysteroid titre and the tissue changes discernible within 6 hr. The bursting firing pattern that is associated with the release of PTTH upon feeding reappears at a time just before the head critical period (ORCHARDand STEEL,1980). Here bursting activity persists for 24 hr
C. G. H. STEELer al.
524
and the ecdysteroid titre begins to increase during this time (STEEL,1982). The coiticidence of this bursting activity and the head critical period was shown by decapitating animals every 4 hr during this period (STEEL,1982); animals decapitated before this period failed to moult. This second period of bursting activity by the medial neurosecretory cells and the subsequent increase in the ecdysteroid titre supports our postulate that this is the second period of PTTH release during larval-adult development in Rhodnius. The above considerations indicate that PTTH release from the brain occurs on two distinct occasions, the first marking the commencement of the plateau phase (at O-2 hr), the second marking the commencement of the large peak (at 5-6 days). However, additional control mechanisms must also exist, for the changes in haemolymph titre following the two releases differ both temporally and quantitatively. The only such mechanism examined in the present paper is the role of excretion. The finding that ecdysteroid excretion in Rhodnius occurs at high haemolymph titres but not at low is the converse of findings with Locusta (HOFFMAN et al., 1974; FEYEREISEN et al., 1976). Since the amounts of ecdysteroid excreted when the titre is increasing (day 10) and decreasing (day 16) are similar, the role of excretion is clearly more subtle than merely the elimination of excess ecdysteroid prior to ecdysis. The observed pattern of ecdysteroid excretion is consistent with the model developed by MADDRELL and GARDINER (1980) to explain the pattern of excretion of other small organic solutes. At times other than diuresis, this model requires selective resorption of such solutes and thereby raises the novel possibility that stage-dependent ecdysteroid excretion may result from control of ecdysteroid resorption from the Malpighian tubule lumen. Acknowledgements-This research was supported by an operating grant from the Natural Sciences and Engineering Council of Canada (A 6669) to C. G. H. Steel, NIH grant (AM-30118) to L. I. Gilbert and NIH (NS-15387) and USDA (59-2167) grants to W. E. Bollenbacher. The expert technical assistance of K. Prasad is gratefully acknowledged.
REFERENCES BOLLENBACHER W. E., VEDECK~S W. V., GILBERTL. I. and O’CONNOR J. D. (1975) Ecdysone titres and prothoracic gland activity during the larval-pupal development of Manduca sexta. Deal. Biol. 44, 4653.
BORSTD. W. and O’CONNORJ. D. (1974) Trace analysis of ecdysones by gas-liquid chromatography, radioimmunoassay and bioassay. Steroids 24, 637-656. BUXTONP. A. (1930) The biology of a blood-sucking bug, Rhodnius prolixus.’ Trans R. eni. Sot. Lond. 78, 227-236.
DEANR. L.. BOLLENBACHER W. E., IAXKE M., SMITHS. L. and GILBERTL. I. (1980) Haemolymph ecdysteroid levels and cellular events in the intermoult/moult sequence of Calpodes ethlius. J. Insect Physiol. 26, 267-280.
FEYEREISEN R., LAGEUXM. and HOFFMANJ. A. (1976) Dynamics of ecdysone metabolism after ingestion and injection in Locusta migratoria. Gen. camp. Endocr. 29, 3 19-327.
GILBERTL. I., GOODMANW. and BOLLENBACHER W. E. (1977) Biochemistry of regulatory lipids and sterols in insects. Int. Rev. Biochem. 14, l-50. GILBERTL. I., BOLLENBACHER W. E., GOODMAN W., SMITH S. L., AGUI N., GRANGER N. and SEDLAKB. J. (1980)
Hormones controlling insect metamorphosis.
Rec. Prog.
Hormone Res. 36, 401449.
GRINGORTEN J. L. and FRIENDW. G. (1979) Haemolymphvolume changes in Rhodnius prolixus during flight. J. exp. Biol. 83, 325-333.
HOFFMANJ. A., KOOLMANJ., KARL~N P. and JOLY P. (1974) Molting hormone titer and metabolic fate of injected ecdysone during the fifth larval instar and in adults of Locusta migratoria (Orthoptera). Gen. camp. Endocr. 22,90-97.
HWANG-HSU K., REDDY G., KUMARANA. K., BOLLENBACHERW. E. and GILBERTL. I. (1979) Correlations between juvenile hormone esterase activity, ecdysone titre and cellular reprogramming in Galleria mellonella. J. Insect Physiol. 25, 105-111. KRUSKALW. H. and WALLISW. A. (1952) The use of ranks in one-criterion variance analysis. J. Am. starist. Ass. 47, 583-621.
LOCKEM. and SMITHD. S. (Eds.) (1980) Insecr Biology in the Future. Academic Press, New York. MADDRELLS. H. P. (1964) Excretion in the blood-sucking bug, Rhodnius prolixus Stil II. The normal course of diuresis and the effect of temperature. J. exp. Biol. 41, 163-176. MADDRELLS. H. P. and GARDINERB. 0. C. (1980) The retention of amino acids in the haemolymph during diuresis in Rhodnius. J. exp. Biol. 87, 315-329. MORRISG. P. and STEELC. G. H. (1975) Ultrastructure of neurosecretory cells in the pars intercerebralis of Rhodnius prolixus (Hemiptera). Tissue Cell 7, 73-90. MORRISG. P. and STEELC. G. H. (1977) Sequence of ultrastructural changes induced by activation in the posterior neurosecretory cells in the brain of Rhodnius prolixus with special reference to the role of lysosomes. Tissue Cell 9, 547-561. ORCHARDI. and STEELC. G. H. (1980) Electrical activity of neurosecretory axons from the brain of Rhodnius prolixus: relation of changes in the pattern of activity to endocrine events during the moulting cycle. Brain Res. 191, 53-65. RIDDIFORDL. M. (1976) Hormonal control of insect epiderma1 cell commitment in uitro. Nature, Lond. 259, 115-1.17. RIDD~FORD L. M. (1978) Ecdysone-induced change in cellular commitment of the epidermis of the tobacco hornworm, Manduca sexta, at the initiation of metamorphosis. Gen. camp. Endocr. 34,438-446. S&H S. L., BOLLENBACHER W. E., CCI~PERD., SCHLEYER H.. WIELGUSJ. J. and GILBERTL. I. (1979) Ecdvsone 2&monooxygenase: characterization of an insect -cytochrome P-450 dependent steroid hydroxylase. Molec. Cell. Endocrin. 15, 111-133. SAL C. G. H. (1978) Nervous and hormonal regulation of, neurosecretory cells in the insect brain. In Comparatiue Endocrinology (Edited by GAILLARDP. J. and BOER H. H. ), pp. 327-330. Elsevier, Amsterdam. STEELC. G. H. (1982) Parameters and timing of synthesis, transport and release of neurosecretion ;n thk insect brain. In Neurosecretion (Edited bv FARNERD. S. and LEDERISK.), pp. 223-233. Plenum Press, New York. STEELC. G. H. and HARMSENR. (1971) Dynamics of the neurosecretory system in the brain of an insect, Rhodnius prolixus, during growth and molting. Gen. camp. Endocr. 17, 125-141.
URIBEC. (1927) On the biology and life history of Rhodnius prolixus Stahl. J. Parasit. 13, 129-136. WIELGUSJ., BOLLENBACHER W. E. and GILBERTL. 1. (1979) Correlations between epidermal DNA synthesis and haemolymph ecdysteroid titre during the last larval instar of the tobacco homworm, Manduca sexta. J. Insect Physiol. 25, 9-16.
WIGGLESWORTH V. B. (1933) The physiology of the cuticle and of ecdysis in Rhodnius prolixus (Triatomidae, Hemi-
Ecdysteroid titre ‘s in Rhodnius ptera); with special reference to the function of the oenocytes and of the dermal glands. Q. J. microsc. Sci. 76, 296318.
WIGGLESWORTH V. B. (1934) The physiology of ecdysis in Rhodnius prolixus (Hemiptera). II. Factors controlling moulting and ‘metamorphosis’. Q. J. microsc. Sci. 77, 191-222. WIGGLESWORTH V. B. (1940) The determination of characters at metamorphosis in Rhodnius prolixus (Hemiptera). J. exp. Biol. 17, 201-222. WIGGLESWORTH V. B. (1952) The thoracic gland in Rhodnius prolixus (Hemiptera) and its role in moulting. J. exp. Biol. 29, 561-570.
525
WIGGLESWORTH V. B. (1957) The action of growth hormones in insects. Symp. Sot. exp. Biol. 11, 204227. WIGGLESWORTH V. B. (1963) The action of moulting hormone and juvenile hormone at the cellular level in Rhodnius prolixus. J. exp. Biol. 40, 231-245. WIGGLESWORTII V. B. (1964) The hormonal regulation of growth and reproduction in insects. Adc. Insect Physiol. 2, 247-336. WIGGLESWORTH V. B. (1970) Structural lipids in the insect cuticle and the function of the oenocytes. Tissue Cell 2, 155-179. WIGGLESWORTH V. B. (1973) The significance of “apolysis” in the moulting of insects. J. Ent. (A). 47, 141-149.