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Neurosecretory control of ecdysone release during puparium formation of flies
CENliR.41, .4ND CORZPAR4TIVIC
ENDOCRINOI.OGY
Neurosecretory
17, 483-489
Control of Ecdysone Release Puparium Formation of Flies’ JAN ZDAREK2
Department
(1971)
of Entomology,
Received
during
G. FRAENKEL
AND
of Illinois,
University
December
Urbana,
Illinois
61801
24, 1970
Larvae of Sarcophaga aryyrostoma fail to pupariate while kept wet, but do so upon transfer to dry conditions after about 28 hr. This period is lengthened to 42 hr after injection of tctrodotoxin, and shortened to 12 hr after injection of ecdysone, independently of whether they are kept wet or dry. It is 12 hr in larvae injected with ecydsone plus tetrodotoxin (which inhibits the puparial contraction) or ecdysone plus a-MDH (which inhibits subsequent tanning). It is rather the lack of inhibitory stimulation arising from a wet surrounding which allows the CNS to activate the ring gland (RG), than a positive stimulation caused by dry conditions. Hind parts of larvae. previously kept wet, were implanted with the CNS-RG complex, or CNS or RG alone, taken from xvct-treated larvae kept dry for various periods. Implantation u-it11 the CNS-RG complex from donors of any age induced a high percentage of pupariation, but it occurred sooner, the older the donors. When implanted with RG alone, the age of the donors was decisive; almost no effect at the time of transfer to the dry, and an increasing effect thereafter. When the donors were prepupae at various periods afler puparium formation the effect decreased with increasing age, but was identical when CNS-RG or RG alone were implanted. CNS alone from donors of any age before or after pupariation had no effect. Before pupariation the rinv D gland becomes increasingly more active while the reverse holds after puparintion. The nervous connection between CNS and RG is important rather as a duct for the neuros:!cretory material, than for its electric:11 charge-carrying properties.
Puparium formation (pupariation) of the cyclorrhaphous flies, though an entirely different process from a regular insect molt, is controlled by the same endocrine events: the neurosecretory system in the brain, reacting to stimuli arising during the maturation process, releases the prothoracotropic hormone to activate the ring gland (RG) which then secretes the molting hormone (ecdysone) . Environmental conditions can modify this course of events. For instance, a sensory input from mechanoreceptors determines the onset of puparium formation ‘This study was supported by NSF grant GB 5441X and NIH grants AI-00533 and 5-KO&GM18,495. ’ Permanent address: Institute of Entomology, Czechoslovak Academy of Sciences, Papircnska 25, Prague.
in larvae of the tsetse fly, Gkossina (Finlayson, 1968). Larvae of several Sarcophaga species do not pupariate when in contact with water, under which circumstances release of ecdysone is inhibited (Ohtaki, 1966; Ohtaki et nZ., 1968; Zdarek and Fraenkel, 1970). It is well known that ecdysone is secreted by the side arms of the RG, a region which is homologous to the prothoracic gland of other insects. Several authors recently drew attention to the presence of an effective ecdysone-inactivating system in fly larvae, and attributed the fluctuations in the ecdysone titer in the hemolymph during puparium formation to the dynamics of this system (Ohtaki et al., 1968; Karlson and Bode, 1969). The question still remains whether or to what extent changes
-is1
ZDAREK
AND
in the ecdysone titer (Shaaya and Karlson, 1965 ; Shayya, 1969) are due to the activity of the inactivating enzyme, or fluctuation; in the activity of the RG, or a combination of both systems. The results presented below show that the activity of the RG in fact changes during the pupariation proccjs in a sense to be expected from the change in the ecdysone titer, and t.hat the activation of ecdysone release by the neurosecretory system decreases toward the end of the larval stage. We also bring new evidence on the nature of the inhibiting influence of wet conditions on the neurosecretory activity of the CNS-RG complex. Md4TERIALS
AND
METHODS
Larvae of Sarcophaga argyrostoma (RobinenuDesvoidy) were used throughout. A convenient method of synchronizing endocrine events in a batch of mature larvae is that devised by Ohtaki (1966). The larvae do not pupariate as long as they are kept in contact with water. This trcatment inhibits any release of ecdysone; this inhibition is relieved upon transfer to dry conditions (Ohtaki et ctl., 1968; Zdarek and Fraenkel, 1970). Mature larvae which had left their food (pork liver) were placed in contact with water for 5 days. When removed from the wet they pupariated 25-30 hr later (Zdarck and Fraenkel, 1970). The larvae were ligated behind the C-US by a heavy duty cotton thread and injected by means of finely drawn glass pipettes, as described elsewhere (Zdarek and Fraenkel, 1969). In the implantation experiments the hosts were the hind parts of larvae which had been kept wrt. They were first immobilized on ice, then cut across behind the central nervous system. These hind parts, which were lacking the CNS-RG complex, received implants of this complex, or the CNS or RG separately, through the open slit. The wound was then closed by a ligature, and further sealed and sterilized by either dipping this region into melted paraffin (when the specimens were to bc kept in the wet), or into talcum powder mixed with penicilin and streptomycin in the ratio 1: 1: 1. As a rule, implants from 3 donors were used for each abdomen. The implanted specimens survived for at least 2 days, during which time the incidents of pupariation were recorded. All dissections of implants were made in Ringer solution as closely as possible before implantation. Pupariation was scored in the usual way, as described elsewhere (Fraenkel and Zdarek, 1970). All experiments
FHAENliEL
were pcrformcd at, about 23’C. The (~dy.;on(~ ~I.VX~ throughout was P-:~c~tlysont: (obtwinrd from S,vntex Corporation. Palo .Uto, California). RESVLTS
Various Treatments Which d,flect the Prepupariation Period (between Treatment and Puparium Fornzation) Mature larvae kept in contact, with water for 5 days were used in all the csperiments listed in Table 1. After the treatment they were returned either to wet (groups 3, 5, 7) or dry conditions (remaining experiments), and the period between treatment and pupariation was recorded. The controls, injected with Ringer, and placed in the dry, pupariated after about 28 hr (group 1). This is supposedly the time needed for the manifestation of a sensory stimulation upon transfer to the dry, plus that needed for activation of the RG by the brain, plus that required for ecdysone to affect the target tissues and to induce pupariation. In order to test this supposition and to learn more about the nature of the water effect and its manifestation the following experiments were designed. Delayed pupariation. Tetrodotoxin was used for its ability to inhibit nervous conduction by specifically blocking sodium conductance. Larvae paralyzed by tetrodotoxin continue developing and pupariate, though with a considerable delay, at about the same time when kept wet and dry (group 2 and 3). Thus, in the absence of information on the wet condition, t,he neuroendocrine mechanism of activating the RG functions normally. In other words, a sensory stimulation by wet inhibits this mechanism. The delay in pupariation is more likely the result of impairment of the general conditions due to the paralysis (respiration, circulation, etc.), than of an interference within the CNS-EtG complex. This interpretation is supported by a different kind of evidence. A temporary ligature was applied tightly behind the CNS and immediately released. The ligature severs all nerves leading to the hind part which consequently becomes paralyzed;
= Between treatment, and puparium b Mean f SE.
formation.
neurohormone, the release of which prethe front part, which cont’ains the CNS-RG complex, remains mobile. Since both parts cedes pupariation (Zdarek and Fraenkel, have a continuous hemocoel, they pupariate 1969). together. These specimens in which the In further experiments wMDH [DL-3greater part of the body was paralyzed (3,4 dihydroxyphenyl) hydrazino-2-methylpupariated with the same delay as those proprionic acid] was used as a specific treated with tetrodotoxin, when kept dry inhibitor of DOPA-decarboxylation (Selig(group 4). Those subsequently kept wet man et al., 1969) ; 10 ,~g per larva inhibits never pupariated (group 5). Obviously the tanning without interfering with puparium information which the CNS still received formation (Fogal and Fraenkel. 1969; from the front end was sufficient to inhibit Bodnaryk, 1970). Ecdysone was injected pupariation. together with wMDH. Inhibition of subAccelerated pupariation. In further ex- sequent tanning in no way affected the periments (&II) precocious pupariation short prepupariation period (cf. groups 6 at about half the time of spontaneous de- and 11). velopment in the dry (group 1) was induced The experiments with injected ecdysone by a massive dose of ecdysone [l.O ,ug showed that all manipulat’ions which greatly /3-ecdysone per abdomen is about 10 times delayed or inhibited pupariation acted the lmit value for this system (Zdarek upon events which precede the release of and Fraenkel, 1970)]. It is noteworthy ecdysone, i.e., the prothoracotropic act,ion that. neither a continuous stay in the wet by the CNS. (group 7) nor application of a temporary The 12-hr prepupariation period induced ligature (group 8) affected this short preby exogenous ecdysone appears to be the pupariation period. Larvae which had been minimum period for ecdysone to affect the injected with ecdysone plus tetrodotoxin receptive tissues to get the overt effect. (group 9)) or ecdysone-injected hind parts Since the spontaneous prepupariation period of permanently ligated larvae (group 10) in the dry is more than twice as long, we showed a slight, but significant delay in conclude that the natural secretion of the pupariation compared with t,hose in groups hormone is not fast enough to induce 6 and 7. This may be explained by the pupariation in the minimum period. absence of the pupariat’ion-accelerating Changes in the activity of the RG, which
486
ZDAREK
a-ill be demonstrated in the following tion, substantiate this contention.
ASI)
sec-
Activity of the CNS-RG Complex before and after Pupariation, and Its Possible Controlling Mechanism The CNS-RG complex, the RG alone or the CNS alone, taken from wet-treated larvae exposed for different periods to the dry, and from prepupae at different times after pupariation, were implanted into hind parts of 0-hr dry larvae as described under Materials and Methods. The rate of pupariation in the hosts is diagrammatically shon-n in Fig. 1. Hind parts implanted with intact (‘NS-RG complex taken from larvae of any age showed a high percentage of pupariation; however, the implants taken from older donors brought about the effect considerably sooner. The ability of the complex taken from prepupae of different ages to induce pupariation decreased with the age of the donors. Implants taken from diapausing, i.e., still older and hormonally inact,ive pupae, gave completely negative results. Puparlatlon % 100
FIZAEXKEL
When the RG alone was implanted the age of the donor was decisive for its ability to induce pupariation. Ring glands from 0-hr old larvae had almost no effect. The effect increased with the age of the larvae, and declined with the age of the prepupae. Implantation of CNS alone gave completely negative results. In order to learn more about the mechanism of neuroendocrine control of pupariation the following 3 additional experiment,s were designed (Table 2). 1. The CNS-RG complex taken from wet-treated larvae was implanted into wet treated abdomens which subsequently rcmaincd in contact with water (Expt. 1). 2. RG and CNS, separated from each other, taken from wet-treated larvae, were implanted together, and the recipients subsequently placed to the dry (Expt. 2). 3. The CNS-RG complex from wet.treated larvae was implanted into isolated abdomens of 0-hr dry larvae, which were simultaneously injected with tetrodotoxin (Expt. 3). The total percentage of pupariation after
Sorcopnogo orgyrostomo3 CNS-RG/obdomen
Hours in the dry
Puporiation % 100
Hoursafter 1 3 RG/abdomen 1
puporiation
7 0
6
I2
Hours in the dry
IS
24
0
6
Hours after
12
IS
24
pupariation
FIG. 1. The effect of implantation of CNS-RG complexes, or RGs alone into hind parts of Sarcophaga bullata larvae on their pupariation. The hosts were ligatured and implanted immediately after a wet-t,reatment. The implants were taken either from larvae kept in the dry for different periods after a wet-treatment, or from prepupae at different times after pupariation. Hatched bars: The hosts pupariated O-24 hours after implantation. Open bars: The hosts pupariated 24-48 hours after pupariation. Numbers of larvae used in each treatment are written next to the columns.
NEUKOSECRETOIIY
CONTftOL
OF
TABLE THE
EFFECT ARDOMENS
OF A WKT
OR I1xr
OF L.\RVAF:
MILII,:U
.UD
PUPARIUAI
2 TJ~TROD~T~XJK
argyrostoma
OF Sarcophaga SEPARATED
ON PUP.\RI.\TJON
CNS-HG
IMPL.\NTI’:D COMPLKP
Jfiliell
x0. of spccimew
\VITH
?;o.
I’xpt.
No.
Implant,,
injection
4si
FOIt.\l.ZTIOS
vivow
THE
OF ISOL.\TJ,:J) IST.KT
Pllpariation
of 5111’-
after 4X hl
OJ~
aftrl
~~ “4 Hl
4S HI
2 y( 2 !z 057
13 c; 10:; ::5 “;
~. CNS-RG connected CNS and RG disconnected CNS-ItG connected; 0.05 pg tet rodotoxin
1 *, ::
n The operation.
implanted
hosts
as well
as the
dowrs
52 4” 37
LVet I )ry 1)l.Y
were
kept
implantation of t’he CNS-RG complex, when kept in the wet, was 43%, as against 72% when kept, dry (Fig. 1). It was 35% in larvae additionally injected with 0.05 tetrodoxin, and kept dry. Whatever the cause for the differences between results in Fig. 1 and Table 2, pupariation percentages of 43 and 35% are still very significantly higher t’han when RG alone (Fig. l), or RG and CNS separately (Table 2) were implanted (7% and lo%, respectively). It can be concluded that the activity of the RG fluctuates. The gland is practically inactive at the beginning of the dry period, when it cannot induce pupariation unless connected with t,lie CNS. Later, it becomes less clcpendent on the CNS and at the time of pupariation it, works autonomously. This stimulating activity of the CNS on the RG was seriously impaired by severing the nervous connection between the two organs, but not by blocking the nervous conduction with tetrodotoxin. This suggests that the nervous connection is important as a duct for the neurosecretory material to pass along the axons of the neurosecretory cells of the brain, rather t,han for its electrical charge-carrying properties (unless conduction in these axons is independent of sodium conductance). DISCUSSION
Our investigations on the effect of implanted RG or CNS-RG complexes generally agree with previous experiments by Vogt and Possom& although we used an
for
5 days
:;,5 “!I :;4
in contact
with
water
preview
to the
entirely different technique, experimental set-up and species of fly. Vogt (1942a,b) induced pupariation in isolated abdomens of Dmsophila hydei by the implantation of ring glands. Glands from the late t,hird stage were more effect’& than those from early third stages. Possomp& (1953) used his newly developed technique of extirpation of the RG from otherwise intact larvae of C. erythrocephaZa to show that in the case of older third instar donors implantation of the RG alone induced pupariation while with younger third instar donors the whole CXS-RG complex was required. Making use of the method of wet-treated larvae, dcviscd by Ohtaki (1966)) we were able to time the physiological ages of both donors and hosts far more accurately thau the previous investigators. By implanting ring glands of different ages we demonstrated that not, only does the RG beromc increasingly niorc artive up to the time of pupariation, but also that this activity, exerted not only by the RG alone but’ also the intact CNS-RG complex, dccreaecs sharply after pupariation and has about disappeared by the time the pul)al molt takcis place inside the puparium. This n-as to be expected from the work of dhaay:~ and Karlson (19651, n-hich showed :I .sllarl) decrease in the ccdpeone titer after pupariation, and that of Fraenkel and Hsiao (1968) on S. n~y~rosto~?zn in pupal diapause where ccdy~one had disappeared by the time of eversion of the head. Significantly the CNS-RG complex from such diapausing pupae was inactive when im-
4%
ZI)AHEIi
,4X1)
planted into isolated hind parts of wettreated larvae. Our experiments shed new light on the nature of the mechanism by which wet conditions inhibit puparium formation in X. peregrina (Ohtaki, 1966) and S. argyrostoma (Zdarek and Fraenkel, 1970). This effect requires a nervous conduction between t’he periphery and the CNS. Larvae treated with tetrodotoxin, which abolishes nervous conduction, do pupariate in the wet’. So do isolated hind parts implanted with a CKS-RG and kept in the wet. Here again no sensory input into the CNS exists. It is rat’her the lack of inhibitory stimulation arising from a wet surrounding which allows the CNS to activate the RG, than a positive stimulation caused by dry conditions. Certainly, contact with water does not prevent pupariation per se by directly inhibiting the target tissues to respond to ecdysone; larvae pupariate in the wet upon injection of massive doses of ecdysone. This also shows that the wet stimulus exerts its effect in the chain of events previous to the release of ecdysone from the gland. We cannot, decide on the basis of our experiments what kinds of receptors are involved in detecting the wet milieu. Are they located externally all over the body, or in a restricted area, or are internal organs also involved? The experiments where a ligation was temporarily applied behind the CNS-RG complex showed that t’he relat,ively small area of the still innervated front end was sufficient for the wet effect to show up. Ostensibly, the state of evacuation of the crop is an important preliminary for the induction of pupariation, together with other maturation processes. In wet-treated larvae, after the food has been evacuated from the crop, there always remains therein a gas bubble which keeps its walls stretched. The bubble disappears after a few hours in the dry. One may speculate that proprioceptors in the wall of the crop, signaling the repletion of the crop, provide the specific stimulation for the CNS to inhibit release of the prothoracotropic hormone. We know now of several neuroendocrine events in insect’ develop-
Flt.\ENIiEL
ment which are regulated by prol)r:occption (see Finlayson, 1968, for review). Distention of the body wall after feeding initiates the molt in Rhodnius (Wigglesworth 1934, 1964). In the pupation of Galleria contraction of the body is a necessary preliminary for the molt (Edwards, 1967) ; prevention of this contraction inhibits this molt by inhibiting secretion of ecdysone (Sehnal and Edwards, 1969 ; Alexander, 1970). REFERENCES
N. J. (1970). A regulatory mechanism of ecdysone release in Gal&u mellonella. J.
ALEXANDER,
Insect BODNARYK,
Physiol. R.
P.
16, 271-276. (1970).
Effect
of
DOPA-de-
carboxylase inhibition on the metabolism of P-alanyl-L-tyrosine during puparium formation in the fleshfly, Snrcophaga bullnta Parker. Comp. Biochem.
Physiol.
35, 221-227.
J. S. (1967). Neural control of metamorphosis in Galletia mellone& (Lepidoptera). J. Insect Physiol. 12, 1423-1433. PINLAYSON, L. H. (1968). Proprioception in the invertebrates. Symp. Zool. Sot. London 23, EDWaRDS.
217-249. W., AND FRAENKEL, G. (1969). The role of bursicon in melanization and endocuticle formation in the adult fleshfly. Sarcophnga bul-
Focal,
lata.
J. Insect
Physiol.
15, 1235-1247.
G., AND HSIAO, C. (1968). Morphological and endocrinological aspects of pupal diapause in a fleshfly, Sarcophaga argyrostoma.
FRAENKEL,
J. Insect
Physiol. 14, 707-718. G., .~ND ZDAREK.
J. (1970). The evaluation of the “Calliphora test” as an assay for ecdysone. Biol. Bull. 139, 138-150. KARLSON. P.. AND BODE, C. (1969). Die Inaktivierung des Ecdysons bei der Schmeissfliege CaZliphora erythrocephaln Meigen. J. Insect Physiol. FRAENKEL.
15,
111-118.
T. (1966). On the delayed pupation of the fleshfly. Sarcophnga peregrinn. Jap. J. Med.
OHT.4K1,
Sci.
Biol.
19,
97-104.
T., MILKMAN, R. P., AND WILLIAMS, C. M. (1968). Dynamirs of ecdysone secretion and action in the fleshfly, Sarcophaga peregrinn,
OHTAKI,
Biol.
Bull.
135,
322-331.
POSSOMP~~S.B. (1953). Recherches exphrimentales sur le dkterminisme de la metamorphose de Calliphora erythrocephala Meig. Arch. Zool. Exp.
Gen.
89, 203-364.
SHAAYA, E. (1969). Der Ecdysontiter wLhrend der Insektenentwickhmg. VI. Untersuchungen iiber die Verteilung in verschiedenen Geweben van
NEUROSECRETORY
CONTROL
Calliphora erythrocephala und iiber seine biolog&he Halbwertszeit. 2. Naturforsch. B 24, 717.-721. SHAAYA, E., AND KARLSON, P. (1965). Der Ecdysontiter wghrend der Insektenentwicklung. II. Die postembryonale Entwicklung der Schmeissfliegc, Calliphora erythrocephala Meig. J.
Insect
Physiol.
11, 6569.
EDWARDS, J. S. (1969). Body constraint and developmental arrest in Galleriu me/lo?zella: Further studies. Biol. Bztll. 137, 352-358. S~LIGXMAN, M., FRIEDMAN, S., AND FRAENKEL, G. (19F9) Bursicon mediation of tyrosine hydroxylntion during tanning of the adult cuticle of the
SEIINAL,
I?.,
AND
fly,
Snrcophnga
&data.
J. Insect
Physiol.
15,
533-561. VOGT. M. (1942a). Die “Puparisierung” als Ringdriisenwirkung. Biol. Ze-ntralbl. 62, 149-154.
OF
PUPARIUM
FORMATION
4x9
VOGT, M. (1942b). Induktion von Metamorllhoseprozessen durch implantierte Ringdriisen bei Drosophila.
Arch.
Entlcricklu?Lgsmecll.
Orgrol.
142, 131-182. V. B. (1934). The physiology of ecdysis in Rhodnius prolizus (Hemiptern). II. Factors controlling moulting and ‘metamorphosis.’ Quart. J. Microsc. Sci. 79, 91-121. WIGGLESWORTTI. V. B. (1964). The hormonal regulation of growth and reproduction in insects. Advan. Insect. Physiol. 2, 246-335. ZD.~R~K, J., ASD FRAENKEL, G. (1969). Correlated effects of ecdysone and neurosecretion in puparium formation (pupariation) of flies. Proc.
WIGGLESWORTH,
Nat.
Acad.
Sci.
U. 8. 64, 565-572.
J., AND FRAENKEL, G. (1970). Overt and covert effects of endogenous and exogenous ecdysone in puparium formation of flies. Proc.
ZDAREK,
Nat.
Aced.
Sri.
CT. S. 67,
331-337.
ENDOCRINOI.OGY
Neurosecretory
17, 483-489
Control of Ecdysone Release Puparium Formation of Flies’ JAN ZDAREK2
Department
(1971)
of Entomology,
Received
during
G. FRAENKEL
AND
of Illinois,
University
December
Urbana,
Illinois
61801
24, 1970
Larvae of Sarcophaga aryyrostoma fail to pupariate while kept wet, but do so upon transfer to dry conditions after about 28 hr. This period is lengthened to 42 hr after injection of tctrodotoxin, and shortened to 12 hr after injection of ecdysone, independently of whether they are kept wet or dry. It is 12 hr in larvae injected with ecydsone plus tetrodotoxin (which inhibits the puparial contraction) or ecdysone plus a-MDH (which inhibits subsequent tanning). It is rather the lack of inhibitory stimulation arising from a wet surrounding which allows the CNS to activate the ring gland (RG), than a positive stimulation caused by dry conditions. Hind parts of larvae. previously kept wet, were implanted with the CNS-RG complex, or CNS or RG alone, taken from xvct-treated larvae kept dry for various periods. Implantation u-it11 the CNS-RG complex from donors of any age induced a high percentage of pupariation, but it occurred sooner, the older the donors. When implanted with RG alone, the age of the donors was decisive; almost no effect at the time of transfer to the dry, and an increasing effect thereafter. When the donors were prepupae at various periods afler puparium formation the effect decreased with increasing age, but was identical when CNS-RG or RG alone were implanted. CNS alone from donors of any age before or after pupariation had no effect. Before pupariation the rinv D gland becomes increasingly more active while the reverse holds after puparintion. The nervous connection between CNS and RG is important rather as a duct for the neuros:!cretory material, than for its electric:11 charge-carrying properties.
Puparium formation (pupariation) of the cyclorrhaphous flies, though an entirely different process from a regular insect molt, is controlled by the same endocrine events: the neurosecretory system in the brain, reacting to stimuli arising during the maturation process, releases the prothoracotropic hormone to activate the ring gland (RG) which then secretes the molting hormone (ecdysone) . Environmental conditions can modify this course of events. For instance, a sensory input from mechanoreceptors determines the onset of puparium formation ‘This study was supported by NSF grant GB 5441X and NIH grants AI-00533 and 5-KO&GM18,495. ’ Permanent address: Institute of Entomology, Czechoslovak Academy of Sciences, Papircnska 25, Prague.
in larvae of the tsetse fly, Gkossina (Finlayson, 1968). Larvae of several Sarcophaga species do not pupariate when in contact with water, under which circumstances release of ecdysone is inhibited (Ohtaki, 1966; Ohtaki et nZ., 1968; Zdarek and Fraenkel, 1970). It is well known that ecdysone is secreted by the side arms of the RG, a region which is homologous to the prothoracic gland of other insects. Several authors recently drew attention to the presence of an effective ecdysone-inactivating system in fly larvae, and attributed the fluctuations in the ecdysone titer in the hemolymph during puparium formation to the dynamics of this system (Ohtaki et al., 1968; Karlson and Bode, 1969). The question still remains whether or to what extent changes
-is1
ZDAREK
AND
in the ecdysone titer (Shaaya and Karlson, 1965 ; Shayya, 1969) are due to the activity of the inactivating enzyme, or fluctuation; in the activity of the RG, or a combination of both systems. The results presented below show that the activity of the RG in fact changes during the pupariation proccjs in a sense to be expected from the change in the ecdysone titer, and t.hat the activation of ecdysone release by the neurosecretory system decreases toward the end of the larval stage. We also bring new evidence on the nature of the inhibiting influence of wet conditions on the neurosecretory activity of the CNS-RG complex. Md4TERIALS
AND
METHODS
Larvae of Sarcophaga argyrostoma (RobinenuDesvoidy) were used throughout. A convenient method of synchronizing endocrine events in a batch of mature larvae is that devised by Ohtaki (1966). The larvae do not pupariate as long as they are kept in contact with water. This trcatment inhibits any release of ecdysone; this inhibition is relieved upon transfer to dry conditions (Ohtaki et ctl., 1968; Zdarek and Fraenkel, 1970). Mature larvae which had left their food (pork liver) were placed in contact with water for 5 days. When removed from the wet they pupariated 25-30 hr later (Zdarck and Fraenkel, 1970). The larvae were ligated behind the C-US by a heavy duty cotton thread and injected by means of finely drawn glass pipettes, as described elsewhere (Zdarek and Fraenkel, 1969). In the implantation experiments the hosts were the hind parts of larvae which had been kept wrt. They were first immobilized on ice, then cut across behind the central nervous system. These hind parts, which were lacking the CNS-RG complex, received implants of this complex, or the CNS or RG separately, through the open slit. The wound was then closed by a ligature, and further sealed and sterilized by either dipping this region into melted paraffin (when the specimens were to bc kept in the wet), or into talcum powder mixed with penicilin and streptomycin in the ratio 1: 1: 1. As a rule, implants from 3 donors were used for each abdomen. The implanted specimens survived for at least 2 days, during which time the incidents of pupariation were recorded. All dissections of implants were made in Ringer solution as closely as possible before implantation. Pupariation was scored in the usual way, as described elsewhere (Fraenkel and Zdarek, 1970). All experiments
FHAENliEL
were pcrformcd at, about 23’C. The (~dy.;on(~ ~I.VX~ throughout was P-:~c~tlysont: (obtwinrd from S,vntex Corporation. Palo .Uto, California). RESVLTS
Various Treatments Which d,flect the Prepupariation Period (between Treatment and Puparium Fornzation) Mature larvae kept in contact, with water for 5 days were used in all the csperiments listed in Table 1. After the treatment they were returned either to wet (groups 3, 5, 7) or dry conditions (remaining experiments), and the period between treatment and pupariation was recorded. The controls, injected with Ringer, and placed in the dry, pupariated after about 28 hr (group 1). This is supposedly the time needed for the manifestation of a sensory stimulation upon transfer to the dry, plus that needed for activation of the RG by the brain, plus that required for ecdysone to affect the target tissues and to induce pupariation. In order to test this supposition and to learn more about the nature of the water effect and its manifestation the following experiments were designed. Delayed pupariation. Tetrodotoxin was used for its ability to inhibit nervous conduction by specifically blocking sodium conductance. Larvae paralyzed by tetrodotoxin continue developing and pupariate, though with a considerable delay, at about the same time when kept wet and dry (group 2 and 3). Thus, in the absence of information on the wet condition, t,he neuroendocrine mechanism of activating the RG functions normally. In other words, a sensory stimulation by wet inhibits this mechanism. The delay in pupariation is more likely the result of impairment of the general conditions due to the paralysis (respiration, circulation, etc.), than of an interference within the CNS-EtG complex. This interpretation is supported by a different kind of evidence. A temporary ligature was applied tightly behind the CNS and immediately released. The ligature severs all nerves leading to the hind part which consequently becomes paralyzed;
= Between treatment, and puparium b Mean f SE.
formation.
neurohormone, the release of which prethe front part, which cont’ains the CNS-RG complex, remains mobile. Since both parts cedes pupariation (Zdarek and Fraenkel, have a continuous hemocoel, they pupariate 1969). together. These specimens in which the In further experiments wMDH [DL-3greater part of the body was paralyzed (3,4 dihydroxyphenyl) hydrazino-2-methylpupariated with the same delay as those proprionic acid] was used as a specific treated with tetrodotoxin, when kept dry inhibitor of DOPA-decarboxylation (Selig(group 4). Those subsequently kept wet man et al., 1969) ; 10 ,~g per larva inhibits never pupariated (group 5). Obviously the tanning without interfering with puparium information which the CNS still received formation (Fogal and Fraenkel. 1969; from the front end was sufficient to inhibit Bodnaryk, 1970). Ecdysone was injected pupariation. together with wMDH. Inhibition of subAccelerated pupariation. In further ex- sequent tanning in no way affected the periments (&II) precocious pupariation short prepupariation period (cf. groups 6 at about half the time of spontaneous de- and 11). velopment in the dry (group 1) was induced The experiments with injected ecdysone by a massive dose of ecdysone [l.O ,ug showed that all manipulat’ions which greatly /3-ecdysone per abdomen is about 10 times delayed or inhibited pupariation acted the lmit value for this system (Zdarek upon events which precede the release of and Fraenkel, 1970)]. It is noteworthy ecdysone, i.e., the prothoracotropic act,ion that. neither a continuous stay in the wet by the CNS. (group 7) nor application of a temporary The 12-hr prepupariation period induced ligature (group 8) affected this short preby exogenous ecdysone appears to be the pupariation period. Larvae which had been minimum period for ecdysone to affect the injected with ecdysone plus tetrodotoxin receptive tissues to get the overt effect. (group 9)) or ecdysone-injected hind parts Since the spontaneous prepupariation period of permanently ligated larvae (group 10) in the dry is more than twice as long, we showed a slight, but significant delay in conclude that the natural secretion of the pupariation compared with t,hose in groups hormone is not fast enough to induce 6 and 7. This may be explained by the pupariation in the minimum period. absence of the pupariat’ion-accelerating Changes in the activity of the RG, which
486
ZDAREK
a-ill be demonstrated in the following tion, substantiate this contention.
ASI)
sec-
Activity of the CNS-RG Complex before and after Pupariation, and Its Possible Controlling Mechanism The CNS-RG complex, the RG alone or the CNS alone, taken from wet-treated larvae exposed for different periods to the dry, and from prepupae at different times after pupariation, were implanted into hind parts of 0-hr dry larvae as described under Materials and Methods. The rate of pupariation in the hosts is diagrammatically shon-n in Fig. 1. Hind parts implanted with intact (‘NS-RG complex taken from larvae of any age showed a high percentage of pupariation; however, the implants taken from older donors brought about the effect considerably sooner. The ability of the complex taken from prepupae of different ages to induce pupariation decreased with the age of the donors. Implants taken from diapausing, i.e., still older and hormonally inact,ive pupae, gave completely negative results. Puparlatlon % 100
FIZAEXKEL
When the RG alone was implanted the age of the donor was decisive for its ability to induce pupariation. Ring glands from 0-hr old larvae had almost no effect. The effect increased with the age of the larvae, and declined with the age of the prepupae. Implantation of CNS alone gave completely negative results. In order to learn more about the mechanism of neuroendocrine control of pupariation the following 3 additional experiment,s were designed (Table 2). 1. The CNS-RG complex taken from wet-treated larvae was implanted into wet treated abdomens which subsequently rcmaincd in contact with water (Expt. 1). 2. RG and CNS, separated from each other, taken from wet-treated larvae, were implanted together, and the recipients subsequently placed to the dry (Expt. 2). 3. The CNS-RG complex from wet.treated larvae was implanted into isolated abdomens of 0-hr dry larvae, which were simultaneously injected with tetrodotoxin (Expt. 3). The total percentage of pupariation after
Sorcopnogo orgyrostomo3 CNS-RG/obdomen
Hours in the dry
Puporiation % 100
Hoursafter 1 3 RG/abdomen 1
puporiation
7 0
6
I2
Hours in the dry
IS
24
0
6
Hours after
12
IS
24
pupariation
FIG. 1. The effect of implantation of CNS-RG complexes, or RGs alone into hind parts of Sarcophaga bullata larvae on their pupariation. The hosts were ligatured and implanted immediately after a wet-t,reatment. The implants were taken either from larvae kept in the dry for different periods after a wet-treatment, or from prepupae at different times after pupariation. Hatched bars: The hosts pupariated O-24 hours after implantation. Open bars: The hosts pupariated 24-48 hours after pupariation. Numbers of larvae used in each treatment are written next to the columns.
NEUKOSECRETOIIY
CONTftOL
OF
TABLE THE
EFFECT ARDOMENS
OF A WKT
OR I1xr
OF L.\RVAF:
MILII,:U
.UD
PUPARIUAI
2 TJ~TROD~T~XJK
argyrostoma
OF Sarcophaga SEPARATED
ON PUP.\RI.\TJON
CNS-HG
IMPL.\NTI’:D COMPLKP
Jfiliell
x0. of spccimew
\VITH
?;o.
I’xpt.
No.
Implant,,
injection
4si
FOIt.\l.ZTIOS
vivow
THE
OF ISOL.\TJ,:J) IST.KT
Pllpariation
of 5111’-
after 4X hl
OJ~
aftrl
~~ “4 Hl
4S HI
2 y( 2 !z 057
13 c; 10:; ::5 “;
~. CNS-RG connected CNS and RG disconnected CNS-ItG connected; 0.05 pg tet rodotoxin
1 *, ::
n The operation.
implanted
hosts
as well
as the
dowrs
52 4” 37
LVet I )ry 1)l.Y
were
kept
implantation of t’he CNS-RG complex, when kept in the wet, was 43%, as against 72% when kept, dry (Fig. 1). It was 35% in larvae additionally injected with 0.05 tetrodoxin, and kept dry. Whatever the cause for the differences between results in Fig. 1 and Table 2, pupariation percentages of 43 and 35% are still very significantly higher t’han when RG alone (Fig. l), or RG and CNS separately (Table 2) were implanted (7% and lo%, respectively). It can be concluded that the activity of the RG fluctuates. The gland is practically inactive at the beginning of the dry period, when it cannot induce pupariation unless connected with t,lie CNS. Later, it becomes less clcpendent on the CNS and at the time of pupariation it, works autonomously. This stimulating activity of the CNS on the RG was seriously impaired by severing the nervous connection between the two organs, but not by blocking the nervous conduction with tetrodotoxin. This suggests that the nervous connection is important as a duct for the neurosecretory material to pass along the axons of the neurosecretory cells of the brain, rather t,han for its electrical charge-carrying properties (unless conduction in these axons is independent of sodium conductance). DISCUSSION
Our investigations on the effect of implanted RG or CNS-RG complexes generally agree with previous experiments by Vogt and Possom& although we used an
for
5 days
:;,5 “!I :;4
in contact
with
water
preview
to the
entirely different technique, experimental set-up and species of fly. Vogt (1942a,b) induced pupariation in isolated abdomens of Dmsophila hydei by the implantation of ring glands. Glands from the late t,hird stage were more effect’& than those from early third stages. Possomp& (1953) used his newly developed technique of extirpation of the RG from otherwise intact larvae of C. erythrocephaZa to show that in the case of older third instar donors implantation of the RG alone induced pupariation while with younger third instar donors the whole CXS-RG complex was required. Making use of the method of wet-treated larvae, dcviscd by Ohtaki (1966)) we were able to time the physiological ages of both donors and hosts far more accurately thau the previous investigators. By implanting ring glands of different ages we demonstrated that not, only does the RG beromc increasingly niorc artive up to the time of pupariation, but also that this activity, exerted not only by the RG alone but’ also the intact CNS-RG complex, dccreaecs sharply after pupariation and has about disappeared by the time the pul)al molt takcis place inside the puparium. This n-as to be expected from the work of dhaay:~ and Karlson (19651, n-hich showed :I .sllarl) decrease in the ccdpeone titer after pupariation, and that of Fraenkel and Hsiao (1968) on S. n~y~rosto~?zn in pupal diapause where ccdy~one had disappeared by the time of eversion of the head. Significantly the CNS-RG complex from such diapausing pupae was inactive when im-
4%
ZI)AHEIi
,4X1)
planted into isolated hind parts of wettreated larvae. Our experiments shed new light on the nature of the mechanism by which wet conditions inhibit puparium formation in X. peregrina (Ohtaki, 1966) and S. argyrostoma (Zdarek and Fraenkel, 1970). This effect requires a nervous conduction between t’he periphery and the CNS. Larvae treated with tetrodotoxin, which abolishes nervous conduction, do pupariate in the wet’. So do isolated hind parts implanted with a CKS-RG and kept in the wet. Here again no sensory input into the CNS exists. It is rat’her the lack of inhibitory stimulation arising from a wet surrounding which allows the CNS to activate the RG, than a positive stimulation caused by dry conditions. Certainly, contact with water does not prevent pupariation per se by directly inhibiting the target tissues to respond to ecdysone; larvae pupariate in the wet upon injection of massive doses of ecdysone. This also shows that the wet stimulus exerts its effect in the chain of events previous to the release of ecdysone from the gland. We cannot, decide on the basis of our experiments what kinds of receptors are involved in detecting the wet milieu. Are they located externally all over the body, or in a restricted area, or are internal organs also involved? The experiments where a ligation was temporarily applied behind the CNS-RG complex showed that t’he relat,ively small area of the still innervated front end was sufficient for the wet effect to show up. Ostensibly, the state of evacuation of the crop is an important preliminary for the induction of pupariation, together with other maturation processes. In wet-treated larvae, after the food has been evacuated from the crop, there always remains therein a gas bubble which keeps its walls stretched. The bubble disappears after a few hours in the dry. One may speculate that proprioceptors in the wall of the crop, signaling the repletion of the crop, provide the specific stimulation for the CNS to inhibit release of the prothoracotropic hormone. We know now of several neuroendocrine events in insect’ develop-
Flt.\ENIiEL
ment which are regulated by prol)r:occption (see Finlayson, 1968, for review). Distention of the body wall after feeding initiates the molt in Rhodnius (Wigglesworth 1934, 1964). In the pupation of Galleria contraction of the body is a necessary preliminary for the molt (Edwards, 1967) ; prevention of this contraction inhibits this molt by inhibiting secretion of ecdysone (Sehnal and Edwards, 1969 ; Alexander, 1970). REFERENCES
N. J. (1970). A regulatory mechanism of ecdysone release in Gal&u mellonella. J.
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J. S. (1967). Neural control of metamorphosis in Galletia mellone& (Lepidoptera). J. Insect Physiol. 12, 1423-1433. PINLAYSON, L. H. (1968). Proprioception in the invertebrates. Symp. Zool. Sot. London 23, EDWaRDS.
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J. (1970). The evaluation of the “Calliphora test” as an assay for ecdysone. Biol. Bull. 139, 138-150. KARLSON. P.. AND BODE, C. (1969). Die Inaktivierung des Ecdysons bei der Schmeissfliege CaZliphora erythrocephaln Meigen. J. Insect Physiol. FRAENKEL.
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EDWARDS, J. S. (1969). Body constraint and developmental arrest in Galleriu me/lo?zella: Further studies. Biol. Bztll. 137, 352-358. S~LIGXMAN, M., FRIEDMAN, S., AND FRAENKEL, G. (19F9) Bursicon mediation of tyrosine hydroxylntion during tanning of the adult cuticle of the
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142, 131-182. V. B. (1934). The physiology of ecdysis in Rhodnius prolizus (Hemiptern). II. Factors controlling moulting and ‘metamorphosis.’ Quart. J. Microsc. Sci. 79, 91-121. WIGGLESWORTTI. V. B. (1964). The hormonal regulation of growth and reproduction in insects. Advan. Insect. Physiol. 2, 246-335. ZD.~R~K, J., ASD FRAENKEL, G. (1969). Correlated effects of ecdysone and neurosecretion in puparium formation (pupariation) of flies. Proc.
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