Japan. J. Med. Sci. Biol., 19, 97-104, 1966

ON THE DELAYED PUPATION OF THE FLESHFLY, PEREGRINA ROBINEAU-DESVOIDY

TETSUYA OHTAKI Department of Medical Entomology, National Institute of Health, Tokyo

(Received : February 19th, 1966)

The larvae of Sarcophaga peregrine do not pupate in a wet condition. If they are placed in a glass vessel containing a certain amount of water, their pupation delays for 100 hr or more. The cause of this delayed pupation is neither a direct action of water to their integument nor disturbance of respiration, because the pupation takes place even in a wet vessel, if they have been previously exposed to a dry situation for a certain period. The mature larvae transferred into a dry vessel from a wet one always pupate 18 to 24 hr later. Ligation experiments show that the hormone inducing the pupation is released 6 hr after transference to a dry condition. When the ligation is made in the middle of the , behind the brain and the ring gland, the hind part of it readily pupates by an injection of ecdysone. The results of these experiments suggest that retardation of ecdysone release is the final reason for the delayed pupation. It seems likely that their removal from water contact induces the secretion of ecdysone from the ring gland.

INTRODUCTION

The larvae of a fleshfly, Sarcophaga peregrina Robineau-Desvoidy, which is one of the most common of medical importance in Japan, usually breed in carrion or in nightsoil pots. When they attain the full size, they cease feeding, leave the breeding site, and then creep into soil where the pupation takes place about one day later. It has been observed, however, that the pupation is delayed for several days when they can not find a favorable site to pupate. This delay of the pupation occurs both with wild flies in the field and with flies bred under experimental condition. If the larvae, reared with hog liver in a glass jar, are not transferred to a drier situation, the pupation never takes place concurrently throughout the same batch. Some of them may remain mature larvae for ten days or more without pupation. It seems probable that the delayed pupation brought by some environmental condition is owing to a possible prevention of hormone release from ring gland, for it has been well established by several investigators that the puparium formation is induced by a hormone which is produced by ring gland. Hence, the following questions may emerge : what is the real factor responsible for this delayed pupation ; is it due to a temporary absence of the hormone secretion ; and how is neuro-endocrine mechanism included in this prevention. Although it has been reported in Lucilia (Cousin, 1932, Evans, 1935, Mellanby, 1938) and in Drosophila (Takaoka, 1960) that delayed pupation may be caused by environmental conditions unfavorable for pupation, a convincing endocrinological

大 滝哲 也(国 立 予 防衛 生研 究所 衛 生昆 虫 部)

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explanation is still lacking to elucidate these phenomena. The present paper deals with a series of experiments which demonstrate retardation of pupation by simple contact with water on sarcophagid species. Studies involving some other species of the are included and a possible mode of action of neuro- endocrine systems on the delayed pupation is discussed.

MATERIALS AND METHODS

Materials used were mature larvae of the fleshfly, Sarcophaga peregrine

Robineau-Desvoidy, which have been routinely maintained in our laboratory. About

five hundred larvae, laid on cotton pads, were transferred to a glass jar (12cm in

diameter and 15cm in height), and reared with 400g of hog liver. After three days,

the larvae had grown to their maximal size, and ceased feeding. The crop of the

larvae gradually became empty and filled with air. In order to get sufficient larvae

of equal maturity for these experiments, they were kept in the same jar for the next

two days, and a certain amount of water was added occasionally to avoid the drying

of their residual food. Under this condition, individuals slow in maturing continued to

develop to full maturation, but none of them pupated. After washing the larval body

with fresh water and draining it on filter paper, the mature larvae were used for the following experiments:

Experiment 1 : To learn the conditions which caused delayed pupation , a number of the larvae were transferred to small glass vessels (9cm in diameter and 6cm in height), which had a filter paper or sand on the bottom. Then, a required amount of water was added to the vessels. After the vessels were tightly covered, some of them were kept under continuous light and the others in complete darkness.

Experiment 2 : The mature larvae were kept in glass vessels containing 5 ml of water for additional 48 hr in order to discard exceptional individuals which early pupated. Then, to know whether the dry condition may induce their pupation, the larvae were transferred to the vessels laid with filter paper alone, for varying period

(1-12 hr). Experiment 3 and 4: Another group of the mature larvae was kept in a wet condition, the same as in Experiment 2, before using. To half of this group ligatures were applied, and injections of ecdysone were given to the remainder. The ligature was made with cotton thread after the method used by Fraenkel (1935). In the ecdysone test, a tight ligature was applied to the mature larvae at the level behind the brain complex. Immediately back of it, a suitable second ligature was made, and the larvae were divided into two pieces. The posterior half was mounted on a glass needle passing through the second ligature, and 0.002ml of water solution of ecdysone was injected in various concentrations. The assay for this test was the same as that of the Calliphora-test, using a quantitative assay of ecdysone which was described by

Karlson (1956).

The rearing of the materials and all experiments were carried out at 25•Ž.

RESULTS

Experiment 1. Delayed Pupation Caused by Simple Contact with Water

The mature larvae were kept in the glass vessels containing various amounts of 1966 DELAYED PUPATION OF FLESHFLY 99 water, 2, 5 and 20ml, and in a vessel to which no water was added for control. After they were transferred to the vessels, the number of individuals beginning pupation was counted every hour for the next 120 hr. These experiments were repeated three times. The results obtained are shown in Fig. 1.

Fig. 1. The pupation time of the mature larvae in the vessels containing 2ml and 5 ml of water, and filter paper alone.

In the dry control*, all larvae pupated during a rather short range of time ; the earliest one started to pupate 18 hr after the transfer. The range of pupation time was a little more extended in continuous light than in darkness. When 2ml of water was poured into the vessel, only filter paper laid on the bottom was wet, and water did not show on the surface. Thus, the body surface of the larvae was not wet except for that portion which was in contact with the filter paper or the glass wall. Even under this condition, the pupation was remarkably delayed, and 50% pupation was not attained within 100 hr. When 5ml of water was poured into the vessel, it rose above the filter paper, and the larval body was entirely wet, but the posterior stigmata were not blocked by the water. Of 30 , only two pupated at 110 hr. The larvae in 20 ml of water were completely submerged in it and could not escape from the water. In this case, none pupated during the period of observation. In similar experiments to know whether dry or wet sand prevents the pupation or not, 50g of dried sand was previously placed into each vessel, and 5 and 20ml, respectively, of water were added. For dry control, vessels to which no water was added were provided. The larvae placed on sand with 5 ml of water burned themselves into the moistened sand, and pupated just above the bottom of vessels. In this case, the major portion of the body surface was not in direct contact with water, and their pupation * The humidity of all vessels was considered the same (saturated) as condensation was appeared on the top of all vessels. 100 OHTAKI Vol. 19 took place almost simultaneously, as shown in Fig. 2.

Fig. 2. The pupation time of the mature larvae in the vessels containing sand and water. In the case of 50 g sand+20 ml of water, no one pupated during the period of observation.

By adding 20ml of water to the sand, a thin layer of water appeared over the sand level, and the larvae could not creep into the sand. The pupation did not occur within 120 hr, showing that the situation was much like that in the vessels with only 5 ml of water. The larvae could not creep into the dry sand either, but most of them pupated in a short period of time.

Experiment 2. Induction of Pupation by Drying When larvae with prolonged pupation, induced by contact with water, were transferred to a dry condition, their pupation took place 18 to 24 hr after transfer. If this retardation of the pupation is only due to a simple cause, such as a direct inhibitory action of water contact on the larval integument, the pupation would be prevented so long as the larvae are in contact with water, irrespective of previous drying. Therefore, a series of experiments was attempted to make sure that larvae, which had been subjected to dry condition for a certain period, could pupate under a wet condition. At first, the mature larvae were kept in the vessels containing 5ml of water for 48 hr, and then they were placed in vessels laid with filter paper alone, for 1, 2, 3, 4, 5, 6, 7, 8, 9 and 12 hr, respectively. After the larvae were returned to the wet vessels, the time, which elapsed from their transfer to dry vessels to the beginning of pupation, was recorded. The results obtained are shown in Fig. 3. Drying for 5 hr or less did not induce pupation of larvae, with the exception of a minimum number of individuals. When drying for 6 hr or more, the pupating rate 1966 DELAYED PUPATION OF FLESHELY 101

Fig. 3. The pupation time of the mature larvae in the wet vessels , after they were exposed to dry condition for 3, 6 and 9 hr. Curves of other drying periods are excluded to avoid complication. In the case of drying for 8 or 12 hr, the pupation time of the larvae was almost equal to that of 9 hr drying. The curve of drying for 7 hr is close to that of 6 hr drying . Drying for 1, 4 and 5 hr did not induce the pupation with the exception of one individual .

rose considerably, even though they were returned to a wet vessel . The range of pupation time gradually shortened as the drying period was extended. The pupation times of the larvae exposed to dry conditions for 8 to 12 hr were much the same as those under dry control of Experiment 1.

Experiment 3. Determination of Timing of Hormone Release Inducing the Pupation By the results of the above experiments, it may be assumed that the secretion of hormones, especially ecdysone, is actually prevented during the period of delayed pupation. Therefore, there is a possibility that the hormone is released only after the larvae have been transferred to a dry condition. To know the timing of hormone secretion, a series of experiments in which a tight ligature was placed between the 7th and 8th segments of the mature larvae was performed . The ligations were made at various time intervals, starting one hr after the transference to dry vessels , through 15 hr. After these treatments, the animals were kept under the dry condition . The results, shown in Fig. 4, are unambiguous . In a one-hour drying period, the pupation did not occur on both parts , anterior and posterior, to ligature in any specimens , showing that there was no sign to indicate hormone release. In accordance with the prolongation of the drying , the i ndividuals which pupated in the anterior part gradually increase in number . In the case of 6-hr drying, the pupation rate of the anterior part exceeded 50% , and one of the 20 tested animals pupated in both part, indicating that the hormones had already circulated through the whole body . By drying for a period of 7 hr or more , the 102 OHTAKI Vol. 19

Fig. 4. The pupation of anterior and posterior parts of ligated larvae after they were placed in the dry condition for various periods.

number of individuals which pupated in both parts increased step by step. It is worthy of note that, if drying is less than 4 hr, the anterior part does not pupate throughout the life, even though they are continuously placed in a dry condition. These results show that there are two critical periods, instead of one which is generally recognized in other : at least 5 hr of drying is necessary for the pupation of the anterior part and several additional hours for the pupation of the whole body. Actual initiation of hormone release may follow a few hours subsequent to the first critical point of this latent period.

Experiment 4. Test of Sensitivity of the Mature Larvae to Ecdysone

In order to verify that a temporary arrest of ecdysone releasing is the final cause of this delayed pupation, ecdysone was injected into the posterior part of ligated

larvae.

One and 6 hr after transfer to a dry condition, the larvae were ligated, and

ecdysone was injected immediately to the hind part of them. Each animal finally

accepted 2.25, 4.5 and 9.0ƒÊg of the crude extract of ecdysone, respectively. For

control, distilled water was injected. Pupation was checked 24 hr after the injection.

The results obtained indicate that the mature larvae possess a high sensitivity to 1966 DELA YED PUPATION OF FLESHFLY 103

Table 1. Sensitivity of hind part of ligated larvae to ecdysone*

* Crude extract of ecdysone obtained from Bombyx mori pupae , and about 1 ƒÊg of it can induce 50% pupation of posterior half of house fly larvae. ecdysone, as shown in Table 1. The majority of the untreated anterior part did not pupate, and posterior half injected with water alone, never pupated. It was, however, observed that the sensitivity was somewhat higher with the animals which were placed under a dry condition for 6 hr, than those for one hr.

Experiment 5. Delayed Pupation Caused by Water Contact in Calli phorid Flies

It was also observed that delayed pupation was caused by water contact on two species of Calliphoridae, Aldrichina grahami (Aldrich) and Calliphora lata Coquillett . These larvae were similarly reared with hog liver, and were kept in their moistened culture for an additional two days after they reached the maximal size. When the mature larvae of these species were placed in the wet vessels , as aforementioned, their pupation was delayed for a period of 100 hr or more ; while the control larvae which were transferred to a dry condition, pupated within 36 hr .

DISCUSSION

It has been demonstrated with ample evidence that in the mature larvae of sarcophagid species, the onset of pupation is delayed by simple contact with water . It can be considered that this delayed pupation is essential to the preservation of the

species, because the pupae will be drowned if pupation occurs in their liquid breeding

site.

This phenomenon is surely different from those observed in Lucilia, of which the

delayed pupation is brought by excessive moisture (Cousin, 1932 , Evans, 1935) or desic- cation (Mellanby, 1938), and in Drosophila, of which the pupation is delayed with

exposure to a high CO2 tension (Takaoka, 1960). In the case of Lucilia , the arrest of development may be prolonged for weeks, if they are returned to a good environment . This prolonged prepupal period in Lucilia, has already been called•hprepupal diapause•h

by these authors, but this term may not be adequate in the case of sarcophagid species, since the prepupal period is prolonged only by the direct action of water contact , and the larvae are continuously moving about for this period. It is also confirmed that 104 OHTAKI Vol. 19

Sarcophaga peregrina pass through the winter with true pupal diapause (Ohtaki). The larvae transferred to a dry condition pupated within 24 hr irrespective of the length of delayed pupation prior to bringing to dryness. This result and those of Experiments 2 and 3 suggest that a neuro-endocrine system is involved in the beginning of delayed pupation, or at its termination. Recently, Fraser (1960) showed, using diapause larvae of Lucilia ceasar, that the thoracic gland were inactive during diapause owing to failure of activation by the brain. Therefore, it seems likely that a stimulation of water contact prevents the brain from releasing an activating factor(s) needed for the secretion of ecdysone from ring gland. The absence of such stimulation will liberate the brain. However, the explanation of necessity of a latent period prior to the secretion of ecdysone requires further experiments. It has been previously reported by Karlson (1956) that, if the Calli phora larvae are reared with liver, no satisfactory results can be obtained in a ecdysone test, since it leads to large variation and to a decrease in readiness to react. At least one reason for this may be attributed to the moistened condition of the culture, where the food remains wet until the larvae develop into their maturity, which has been observed in our laboratory. When the other food material, e. g. horse heart, is given, the culture usually gets drier at the end of rearing, and the pupation occurs in a short range of time. If the larvae are bred in liver, it is necessary for getting a number of synchronously developed larvae that one may remove them from the culture to wet vessels and keep them there for 2 days after they reach full maturation. Six-hour drying period following these treatments will successfully induce the pupation of the anterior half in more than 50% of the ligated larvae. The posterior half of them which did not pupate possess a high sensitivity to ecdysone, suggesting that it can be used as a material for the ecdysone test.

The author wishes to express his hearty thanks to Dr. S. Asahina, chief of the Department of Medical Entomology, National Institute of Health, for his valuable advice and encouragement. Cordial thanks are also expressed to Dr. M. Kobayashi, Sericultural Experiment Station, for kind supply of ecdysone.

REFERENCES

COUSIN, G. (1932): Etude experimentale de la diapause des insectes. Bull. Biol. Fr. Belg., Suppl. 15, 1-341. EVANS, A. C. (1935): Studies on the influence of the environment on the sheep blow-fly, Lucilia sericata Meig. II. The influence of humidity and temperature on prepupae and pupae. Para- sitology, 27, 291-298. FRAENKEL, G. S. (1935): A hormone causing pupation in the blowfly Calliphora erythrocephala. Proc. Roy. Soc. London, B. 118, 1-12. FRASER, A. (1960): Humoral control of metamorphosis and diapause in the larvae of certain Cal- liphoridae (Diptera: Cyclorrhapha). Proc. Roy. Soc. Edin., B. 67, 127-140. KARLSON, P. (1956): Biochemical studies on hormones. Vitamins and Hormones, 14, 227- 266. MELLANBY, K. (1938): Daapause and metamorphosis of the blow fly, Lucilia sericata Meig. Parasitology, 30, 392-402. OHTAKI, T. (unpublished data). TAKAOKA, M. (1960): Studies of the metamorphosis in insects IV. Inhibition of pupation by carbon dioxide in the mature larvae of Drosophila melanogaster. Embryologia, 5, 74-84.