The effects of 20-hydroxyecdysone on haemolymph pressure pulsations in Tenebrio molitor

0022-1910184 $3.00+O.OO

J. /n.rr~~ Phwiol.Vol. 30.No. IO.pp. 797-802.1984 Printedin Great Britain. All rights reserved

Copyright < 1984Pergamon Press Ltd

THE EFFECTS OF 20-HYDROXYECDYSONE HAEMOLYMPH PRESSURE PULSATIONS TENEBRZO

ON IN

MOLZTOR

ROBERT FARKAS Institute of Experimental Biology and Ecology. Slovak Academy of Sciences, Sienkiewiczova I. 81434 Bratislava. Czechoslovakia (Received 2 February 1984; revised 28 March 1984)

Abstract-Changes in haemocoelic pressure have been studied after the injection of exogenous 20-hydroxyecdysone, using a special tensometric method. Application of the hormone before the endogenous peak of ecdysteroid causes an acceleration of the progressive changes in the pulsation pattern. When given during the endogenous ecdysteroid peak, 20-hydroxyecdysone produces a retention of the existing type of pulsation. Also, administration of the hormone after the endogenous peak induces a retardation in the developmental programme of the pulsations. Shortly before ecdysis, the exogenous hormone does not affect the pulsation programme or the ecdysis. These changes may represent an elegant involvement of example of a homeostatic function of ecdysteroids in insect development. 20-hydroxyecdysone in regulation of the basic haemolymph pressure is discussed. Key Word

Zndex:

Haemocoelic pulsations. homeostatic regulation, developmental programming

INTRODUCTION It has been found (Slbma, 1976) that the stationary metamorphosis stages of some Endopterygote insects exhibit regular pulsations in haemolymph pressure. The pulsations show a constant pattern which undergoes a characteristic variation with respect to developmental changes in the body. The course of changes in haemolymph pressure is further modified by the effects of temperature, relative humidity and by some other environmental factors (Provansal el al., 1977). Recording these regular haemolymph pressure pulsations appears thus to be a convenient tool for monitoring developmental changes, and the effects of drugs or pathogens (Slima and Lysenko, 1981). In relation to the assumed homeostatic function of ecdysteroids in insect development, Sl6ma (1980) has found that injection of exogenous 20-hydroxyecdysone disturbed the normal pattern of haemolymph pressure before adult ecdysis. The pattern of the pulsations, indicating advanced stages of the ecdysial process, is reversed to show pulsations that are otherwise characteristic of the earlier stages. Since the described developmental changes in haemolymph pressure pulsations are rather constant and accurately reflect the realization of the morphogenetic programme, I have now injected 20-hydroxyecdysone into the body before, during, and after the peak of ecdysteroids.

When it was necessary, the precise physiological age of the pre-pupae and pupae was estimated according to the progressive pigmentation of the respective pharate structures (Wigglesworth, 1948; StellwaagKittler, 1954; Delachambre, 1970). The methods for measurement of haemolymph pressure have been described by Sllma (1976). The insect haemocoele was linked to a Ringer-filled hydraulic transducer system through a thin stainless steel hypodermic needle. The hydraulic transducer system was separated from the haemolymph by a I ~1 droplet of light mineral oil. 20-Hydroxyecdysone (Rohto Pharmaceutical Co. Ltd, Osaka, Japan) was injected in 1 ~1 of 10% ethanolic Ringer solution. The final doses, as revealed by preliminary testing. were 0.5 pg (small dose), 1.O and 2.0 pg (large dose) for each insect. The controls were injected with lo?; ethanolic Ringer alone. The solutions to be tested were injected into the body through the measuring needle after 1-3 h of control recording to reveal the normal haemolymph pressure pattern. The values of haemolymph pressure are given in Pascal (Pa) units relative to the local atmospheric pressure (1 mm of hydrostatic pressure is approx 10 Pa). The results are based on comparative analysis of 155 individual (,I IO experimental and 45 control) tensometric records of which only a few have been selected for illustration. The separate characteristic measures of haemolymph pressure pulsations are explained in Fig. 1B.

MATERIALS AND METHODS

Yellow mealworms, Tenebrio molitor L. was reared on Larsen’s artificial diet and drinking water, kept at 24°C and under 14 h light each day. The pupae used for the experiments were collected daily from the stock of immobile pre-pupae. Under the above described conditions pupal development took 8 days.

RESULTS pupal-adult interecdysial period in the constant, developmentally linked changes in haemolymph pressure have been described as follows (see Provansal et al., 1977): (a) the baseline pressure decreases regularly from + 500 to - 200 or During

Tenebrio,

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the

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FAKKAS

- 500 Pa towards the middle of the period and then it rises again to reach values over 1 kPa before ecdysis; (b) the rhythmic pulsations are individual or paired at the beginning, they are grouped into series of 8-10 pulses intercalated with long periods of rest at the middle of interecdysial period, while their frequency increases continuously to show a constant pulsation pattern before ecdysis. Similar developmental changes in haemolymph pressure have been found recently in the pharate pupal period also and

during the pupal ecdysis. For more details see FarkaS (1983). The pattern of developmentally normal pulses in pre-pupa and pupa are summarized in Fig. I. The pharate pupae and adults are slightly more sensitive to the control injection of Ringer. Their basic haemolymph pressure may increase temporarily for 0.220.5 kPa over the original pressure 20-30 min after the injection. Short-term changes in the amplitude or frequency are very rare. 20-Hydroxyecdysone, IO h after injection into

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Patterns of haemolymph pressure pulsations during characteristic developmental periods. A. Pulsation pattern of pre-pupa 26-24 h before pupal ecdysis. Note the change of continual pulsation to pulse series formation. B. Single or, more commonly, a double pulse separated by long resting period typical for pattern of young pupa 7-10 h after ecdysis. Characteristic measures of pressure pulsations: I. maximal amplitude. 2. real amplitude of the small contractions, 3. pulsation activity indicating percentage of total time spent in pulses, 4. level of the basic pressure during the resting period, 5. resting period. C. Haemolymph pulsations in 4-day old pupa. The pulse series is composed of 8-15 individual pulses. Basic pressure shows sub-atmospheric level. D. Pumping pattern of pharate adult characterized by intensive pulsation activity accompanied by dorsoventral movements of abdomen.

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Fig. 2. The series of several

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pulses and subatmospheric basic pressure 20-hydroxyecdysone in l-day pupa.

freshly ecdysed pupae, causes premature acceleration of the pulsation pattern that is similar to the 34-day old pupa. This is characterized by a switch from individual pulses to a series of several pulses (Fig. 2). About 15-20 h after hormone injection the real amplitude of the contractions and the total time spent in pulses approach the levels that are normally found in a 4-day old pupa, i.e. at the time of the ecdysteroid peak in the body. The basic pressure decreases below the atmospheric level. In this case, the accelerated pattern either reverts to the normal pattern of pulsation and then pupa exhibits the normal developmental progression of pulses or it is retained up to the 4th day. Ecdysis of these animals is delayed for 24-48 h as revealed from g-day permanent records. On the other hand, young pupae cannot overcome the effects of 1 or 2 fig doses of the hormone and the pulsations are, in this case, irreversibly disturbed.

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Fig. 3. Preserved

pulsation

and basic pressure

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Similar responses to the hormone as described above have been obtained with the same treatment given on the 2nd or 3rd day of pupal development. These results have been obtained in 17 cases out of 23 records made. During normal development, the pulsation pattern of a 4-5-day pupa progresses slowly towards the 6th day pattern. This is characterized by a successive elevation of the basic pressure from the subatmospheric levels of equilibrium with the atmospheric pressure, and further by continuous increase of the total pulsation activity. These progressive changes in haemolymph pressure are disturbed considerably by the exogenous 20-hydroxyecdysone. The pattern of the 4-5-day old pupae has been in this case retained for 2-3 days. The selected record in Fig. 3 illustrates the described retention of the old pulsation pattern as well as the retention of sub-atmospheric

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patterns 24 h after 20-hydroxyecdysone old pupa.

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ROBERT FARKAS

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Fig. 4. Gradual disturbance of pumping pattern and pulsation after 20-hydroxy%dysone injection (arrow) into pharate adult. Rapid decrease of basic pressure and of total pulsation activity were induced with 2 pg of the hormone.

basic pressure in a 6-day old pupa which was injected with the hormone 24 h before. The described phenomenon was observed in 13 out of 17 cases. These specimens showed slightly delayed adult ecdysis with more or less normal peristaltic movements. In the pharate adults during the 6th to 8th days of pupal development, l-3 h after the injection of the hormone the frequency and amplitude of the pulsations sharply decreased. Moreover, after 2 additional hours, the haemolymph pressure pulsations resembled the type normally found in the middle of the inter-ecdysial period. Thus, the basic pressure decreased from 750 or 1000 Pa down to atmospheric pressure. During the next 5 h the pulsations were completely abolished, as can be seen in Fig. 4. These effects mostly resulted in death of the injected animals (22 dead out of 26 specimens measured).

In order to compare the effects of 20-hydroxyecdysone between the pharate adults and pharate pupae, I have also injected the immobile prepupae with the hormone and measured the changes in haemolymph pressure throughout pupal ecdysis. Five hours following the injection of 1 pg of hormone into the immobile prepupae (approx 30 h before pupal ecdysis) the relationships between the maximum and real amplitude of the small contractions have been clearly disturbed. This is characterized especially by irregularities in the maintenance of the basic pressure. Formation of abnormal pulsation series and of the prolonged resting periods in these experimental animals are shown in Fig. 5. About 10 h after the injection of 20-hydroxyecdysone the basic pressure and the real amplitude have considerably diminished. Furthermore, about 20 h after

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Fig. 5. Abnormal pulse series and irregular basic pressure in pre-pupa which was injected with I pg of 20-hydroxyecdysone 8 h previously.

the injection of the hormone, the real amplitude of the pulsations has become completely irregular and the pulsations appear as irregular peaks. These observations were made on 23 specimens from 25. Application of 20-hydroxyecdysone less than 8-10 h before the pupal or adult ecdysis has no or little effect on realization of the ecdysial programme. The differences between the doses of 0.5, 1 and 2pg of the hormone injected were negligible. Rarely it was possible to observe small, non-coordinated, changes of the real amplitude and a small decline of the basic pressure. Within the mentioned period of 8-10 h the peristaltic movements associated with ecdysis appeared independent of the presence of the exogenous hormone. However, the effects may appear later, i.e. after the pupal ecdysis. They are manifested by acceleration of the pulsation pattern and premature appearance of the pattern characteristic for the 4-day pupa. This was observed in 15 out of 19 injected specimens.

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DISCUSSION

Realization of the pupal and then adult developmental programmes in Tenebrio is associated with two large peaks in the endogenous titre of ecdysteroids: one occuring in the pre-pupa about 50 h before pupal ecdysis and the other in 4-day old pupa (Delbecque et al., 1978). It thus appears that exogenous supply of 20-hydroxyecdysone outside these periods of ecdysteroid peaks would cause desynchronization of the constant pulsation programme as revealed by the records of haemolymph pressure. According to the described results, we observe several kinds of responses in the haemolymph pressure to the hormone: (a) premature induction of the pulsation associated with future developmental programme; (b) retention of the existing pulsation pattern, and (c) retardation of the realization of future pulsation programme. The succession of the developmental cycles, including special physiological

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Fig. 6. Progressive disturbance of pulsation in the same pre-pupa

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802

ROBERT FARKAS

functions. appears to be controlled by a hierarchy of reactions representing a homeostatic system (Wigglesworth, 1964). It thus appears that the abovementioned experimental data represent a good examof such a homeostatic regulation of ple developmental processes by 20-hydroxyecdysone, as has been suggested by Y&ma (1980). The ecdysteroid-induced acceleration of the pulsation pattern in young pupae is less pronounced than the rapid retardation of the progressive changes in the pattern of pharate adults. Elimination of the hormone effects in young pupae is accompanied by a gradual equilibration of basic pressure to the level normal for the 4th day of pupal life. when the endogenous peak of ecdysteroids synchronizes developmental processes (Slima, 1980). Pharate adults can not eliminate effective doses of the hormone, presumably, because there is no further synchronization ecdysteroid peak in adult development and thus. the pulsation pattern is irreversibly disturbed. The duration of the arrested pulsation programme in the middle-aged pupa depends on the dose of the injected hormone. The normal pattern of pulsation is resumed after disappearance of the hormone effects. This fact suggests that progressive development of ecdysial symptoms is connected in sirlc with declining ecdysteroid peak. On the other hand, retardation of the pulsation programme is due to a repeat of the situation when the endogenous peak was present. This confirms the observations of Slima (1980) who came to the conclusion that 20-hydroxyecdysone can not only prevent the development of ecdysial symptoms, but it can temporarily revert the function of the neuromuscular mechanism back to the previous stage. With reference to the U-shaped curve of respiratory metabolism in Tenebrio pupae (Provansal et al., 1977) and its coincidence with pressure pattern. the endogenous ecdysteroid peak occurs at the minimal basic pressure. 20-Hydroxyecdysone application before as well as after endogenous peak causes decrease of the basic pressure towards and below 200. Simultaneously, the frequency and amplitude of pulses are also affected by the hormone. According to these facts, the changes in pulsation induced by the

hormone may significantly reflect changes in basic processes such as gaseous exchange, water economy and osmotic imbalance. Therefore, it is very likely that 20-hydroxyecdysone may regulate the neurohormones which are generally known to influence the permeability of cell membranes, salt concentration, tone of muscles etc. (Slrima et al., 1974). AciC-nowledgements-I am extremely grateful to Dr Karel Slima. Institute of Entomology. Czechoslovak Academy of Sciences, Prague for his helpful and critical comments during preparation of the manuscript. Part of these results were presented at the 6th European Ecdysone Workshop in August 1983 in Szeged, Hungary.

REFERENCES Delachambre J. (1970) Etudes sur I’tpicuticule des insectes. 1. Le diiveloppement de I’tpicuticule chez l’adulte de Tenehrio molitor. 2. Zellforsch. 108, 38C-396.

Delbecque J.-P., Hirn M., Delachambre J. and De Reggi M. (I 978) Cuticular cycle and moulting hormone levels during the metamorphosis of Tenebrio molitor. Ded Biol. 64, 1l-30. FarkaS R. (1983) Changes in haemolymph pressure pulsations during prepupal development and pupal ecdysis in Tenebrio molifor. Acta em. bohemosl. So, 177-183.

Provansal A., Baudry-Partiaoglou N. and Slama K. (1977) Haemolymph pressure pulses in the metamorphosis of Tenebrio mofitor. Acta ent. bohemosl. 74, 362-374.

Slima K. (1976) Insect haemolymph pressure and its determination. Acta cnl. bohemosl. 13, 65-75. Slima K. (1980) Homeostatic function of ecdysteroids in ecdysis and oviposition. Acfa enr. bohemosl. 77, 145-168. Slama K. and Lysenko 0. (198 I) Monitoring the course of bacterial infections by haemolymph pressure pulses in insects. J. Invert. Path. 37, 1I-21. Slama K., Romariuk M. and Sorm F. (1974) Insecf Hormones and 5iounalogue.s. Springer, Berlin. Stellwaag-Kittler F. (1954) Zur Physiologie der Ktiferhlutung Untersuchungen am Mehlklfer Tenehrio moliror L. Biol. Zb. 13, 12-49. Wigglesworth V. B. (1948) The structure and deposition of the cuticle in the adult mealworm Tenrbrio moliror L. Q. Jl micrsc. Sci. 89, 197-217.

Wigglesworth V. B. (1964) Homeostasis in insect growth. Symp. Sot.

exp. Bid.

18, 265-281.